Methods of screening disorders related to apoE

ABSTRACT

The present invention provides methods inhibiting formation of neurofibrillary tangles; and methods for treating disorders relating to apolipoprotein E (apoE) in a subject. The methods generally involve reducing the level of a carboxyl-terminal truncated form of apoE in a neuronal cell of a subject. The invention further provides isolated cells comprising a nucleic acid molecule encoding a carboxyl-terminal truncated form of apoE; and methods of screening compounds using the cells. The invention further provides compounds that inhibit an apoE cleavage enzyme, and that reduce the formation of neurofibrillary tangles in a neuronal cell. The invention further provides transgenic non-human animals that include as a transgene a nucleic acid that encodes a carboxyl-terminal truncated form of apoE; as well as methods of screening compounds using transgenic animals.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 60/245,737, filed Nov. 3, 2000, which application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to apolipoprotein E, and in particular to methodsof treating disorders relating to ApoE.

BACKGROUND OF THE INVENTION

Human apolipoprotein (apo) E has three major isoforms, apoE2, apoE3, andapoE4 (for review see Mahley and Huang (1999) Curr. Opin. Lipidol.10:207-217). It has been established that apoE4 is associated withincreased plasma cholesterol levels and higher risk for the developmentof coronary heart disease. Dallongeville (1992) J. Lipid Res.33:447-454. ApoE4 has also been linked to the pathogenesis ofAlzheimer's disease. The apoE4 allele is a major risk factor orsusceptibility gene associated with approximately 40-65% of cases ofsporadic and familial Alzheimer's disease and it increases theoccurrence and lowers the age of onset of the disease. Corder et al.(1993) Science 261:921-923. In addition, the apoE4 allele is alsoassociated with poor clinical outcome in patients with acute head traumaand stroke. Slooter et al. (1997) JAMA 277:818-821; and Nicoll et al.(1996) Neuropathol. Appl. Neurobiol. 22:515-517.

The neuropathological hallmarks of Alzheimer's disease are the presenceof neuritic amyloid plaques and neurofibrillary tangles in the brain.Selkoe (1991) Neuron 6:487-498; and Roses, et al. (1994) Curr. OpinionBiotechnol. 5:663-667. The neuritic plaques represent extracellulardeposits of amyloid. The major component of the deposits is the amyloidbeta (Aβ) peptide (DS487), which is the proteolytic product of theamyloid precursor protein (APP). In contrast to amyloid plaques,neurofibrillary tangles are primarily intracellular deposits composedlargely of highly phosphorylated microtubule-associated protein, tau(p-tau), and, to a lesser extent, phosphorylated NF-H (p-NF-H). ApoE isfound in both amyloid plaques and the neurofibrillary tangles

There are many hypotheses to explain the association of apoE4 allelewith the development of Alzheimer's disease, including its modulation ofamyloid β (Aβ) deposition or clearance in the brain, and a lack ofinteraction of apoE4 with the microtubule-associated protein, tau, whichbinds to and stabilizes microtubules.

Alzheimer's disease is an insidious and progressive neurologicaldisorder for which there is currently no cure. In view of the lack ofadequate treatment for Alzheimer's disease, there exists a need fornovel treatment methods for this neurological disorder. The instantinvention provides methods of treating disorders relating to ApoE4, andmethods of reducing neurofibrillary tangles associated with Alzheimer'sdisease.

Literature

Huang et al. (2001) Proc. Natl. Acad. Sci. USA 98:8838-8843; U.S. Pat.No. 6,046,381.

SUMMARY OF THE INVENTION

The present invention provides methods inhibiting formation ofneurofibrillary tangles; and methods for treating disorders relating toapolipoprotein E (apoE) in a subject. The methods generally involvereducing the level of a carboxyl-terminal truncated form of apoE in aneuronal cell of a subject. The invention further provides isolatedcells comprising a nucleic acid molecule encoding a carboxyl-terminaltruncated form of apoE; and methods of screening compounds using thecells. The invention further provides compounds that inhibit an apoEcleavage enzyme, and that reduce the formation of neurofibrillarytangles in a neuronal cell. The invention further provides transgenicnon-human animals that include as a transgene a nucleic acid thatencodes a carboxyl-terminal truncated form of apoE; as well as methodsof screening compounds using transgenic animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the effect of Aβ1-42 on formation ofintracellular inclusions in Neuro-2A cells producing apoE.

FIG. 2 is a graph depicting the percentage ofGFP-apoE3(Δ272-299)-transfected and GFP-apoE4(Δ272-299)-transfectedcells that contain neurofibrillary tangles.

FIGS. 3A-C depict the structure of apoE (FIG. 3A); results indicatingamino acids of apoE that interact with p-tau and p-NF-H (FIG. 3B); andresults indicating the role of the amino terminal domain of apoE information of neurofibrillary tangles (FIG. 3C).

FIG. 4 depicts protein blots of brain lysates from normal individuals(N1 and N2) and individuals with Alzheimer's disease (AD1, AD2, andAD3), immunoprecipitated with anti-apoE antibody.

FIG. 5 is a graph depicting carboxyl-terminal truncated apoE3 and apoE4in brains of NSE-apoE3 and NSE-apoE4 mice.

FIG. 6 is a graph depicting age-dependent accumulation of p-tau inbrains of NSE-apoE mice.

FIG. 7 is a graph depicting the occurrence of p-tau-positiveintraneuronal inclusions in the hippocampus of NSE-apoE4 transgenicmice.

FIG. 8 is a graph depicting the effect of various agents on theenzymatic activity of an enzyme that catalyzes proteolytic cleavage ofapoE.

FIG. 9 is a graph depicting the results of inhibition of an apoEcleaving enzyme by various peptides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the observation that carboxyl-terminaltruncated apoE induces formation of neurofibrillary tangle-likestructures in cultured neurons. Truncation of up to 30 amino acids fromthe carboxyl terminus of apoE results in intracellular inclusions incultured neuronal cells. The inventors found that C-terminal truncatedforms of apoE, but not full length apoE, interact with thephosphorylated form of the microtubule associated protein p-tau and thephosphorylated neurofilament protein of high molecular weight, p-NF-H,and induce neurofibrillary tangle (NFT)-like structures in cells. Thiseffect is specific to neuronal cells.

The NFT-like structures induced by C-terminal truncated apoE are similarin many aspects to NFT seen in the brain of Alzheimer's disease (AD)patients. First, the formation of NFT-like structures induced byC-terminal truncated apoE is neuron-specific. Second, the NFT-likestructures contain p-tau, which is a major component of NFT in ADbrains, and pNF-H, which is also found in NFT in AD brains. Third,electron microscopy reveals that the NFT-like structures induced bytruncated apoE are composed of many irregular filaments, with diametersof 10-20 nm, which are similar to some NFT seen in human AD brains.

The present invention provides methods of inhibiting formation ofneurofibrillary tangles; and methods for treating disorders relating toapolipoprotein E (apoE) in a subject. The methods generally involvereducing the level of a carboxyl-terminal (C-terminal) truncated form ofapoE in a neuronal cell of a subject and/or blocking the interaction ofthe carboxyl-terminal truncated apoE with p-tau and/or p-NF-H.

The invention further provides isolated cells that include a nucleicacid encoding a C-terminal truncated form of apoE. The subject cells areuseful for screening compounds that block the interaction of thecarboxyl-terminal truncated apoE with p-tau and/or p-NF-H, and whichtherefore reduce formation of neurofibrillary tangles.

The invention further provides in vitro screening methods foridentifying compounds that reduce the formation of carboxyl-terminaltruncated apoE and/or that block the interaction of thecarboxyl-terminal truncated apoE with p-tau and/or p-NF-H in a cell. Invitro screening methods for identifying compounds that reduce theformation of carboxyl-terminal truncated apoE involve the use of hostcells that produce full-length apoE that is subsequently proteolyticallyprocessed to carboxyl-terminal truncated apoE. In vitro screeningmethods for identifying compounds that block the interaction of thecarboxyl-terminal truncated apoE with p-tau and/or p-NF-H generallyinvolve the use of subject host cells that include a nucleic acidencoding a C-terminal truncated form of apoE. Compounds identified usingan in vitro screening assay of the invention are candidate compounds fortreating disorders arising from the presence in a neuronal cell ofC-terminal truncated forms of apoE, including AD.

The invention further provides transgenic non-human animals that includeas a transgene a nucleic acid that encodes a carboxyl-terminal truncatedform of ApoE. The subject non-human transgenic animals are useful forscreening compounds that block the interaction of the carboxyl-terminaltruncated apoE with p-tau and/or p-NF-H.

The invention further provides in vivo screening methods for identifyingcompounds that reduce the formation of carboxyl-terminal truncated apoEand/or that block the interaction of the carboxyl-terminal truncatedapoE with p-tau and/or p-NF-H in a cell. In vivo screening methods foridentifying compounds that reduce the formation of carboxyl-terminaltruncated apoE involve the use of transgenic non-human animals thatinclude as a transgene a nucleic acid that encodes full-length apoE, andthat produce, in neuronal cells, full-length apoE that is subsequentlyproteolytically processed to carboxyl-terminal truncated apoE. In vivoscreening methods for identifying compounds that block the interactionof the carboxyl-terminal truncated apoE with p-tau and/or p-NF-Hgenerally involve the use of subject transgenic non-human animals thatinclude as a transgene a nucleic acid that encodes a carboxyl-terminaltruncated form of apoE, and that produce, in neuronal cells, acarboxyl-terminal truncated apoE. Compounds identified using an in vivoscreening assay of the invention are candidate compounds for treatingdisorders arising from the presence in a neuronal cell of C-terminaltruncated forms of apoE, including AD.

The invention further provides compounds that inhibit an apoE cleavageenzyme, and compositions, e.g., pharmaceutical compositions, thatinclude the compounds. The compounds are useful for reducing the levelof neurofibrillary tangles in a neuronal cell, and thus are useful fortreating disorders such as AD.

DEFINITIONS

As used herein, the terms “a disorder associated with apoE,” “a disorderrelated to carboxyl-terminal truncated apoE” and “a disease associatedwith carboxyl-terminal truncated apoE” are used interchangeably to referto any disease or disorder which is associated with the presence in anindividual of carboxyl-terminal truncated apoE. A disease or disorderrelated to carboxyl-terminal truncated apoE may be a direct or indirectresult of the presence of carboxyl-terminal truncated apoE in theindividual. For example, a disease or disorder related tocarboxyl-terminal truncated apoE is one that occurs as a result of theformation of neurofibrillary tangles in a neuronal cell. A disease ordisorder related to carboxyl-terminal truncated apoE may also be adisease or disorder for which carboxyl-terminal truncated apoE is a riskfactor, e.g., the presence in an individual of carboxyl-terminaltruncated apoE increases the individual's risk of developing the diseaseor disorder. Diseases and disorders associated with carboxyl-terminaltruncated apoE include, but are not limited to sporadic Alzheimer'sdisease; familial Alzheimer's disease; diseases associated withincreased plasma levels of cholesterol, such as coronary artery disease;and poor clinical outcome in patients with acute head trauma; and poorclinical outcome in patients with stroke.

The term “Alzheimer's disease” (abbreviated herein as “AD”) as usedherein refers to a condition associated with formation of neuriticplaques comprising amyloid β protein primarily in the hippocampus andcerebral cortex, as well as impairment in both learning and memory. “AD”as used herein is meant to encompass both AD as well as AD-typepathologies.

The term “phenomenon associated with Alzheimer's disease” as used hereinrefers to a structural, molecular, or functional event associated withAD, particularly such an event that is readily assessable in an animalmodel. Such events include, but are not limited to, amyloid deposition,neuropathological developments, learning and memory deficits, and otherAD-associated characteristics.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably herein to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes single-, double-stranded andtriple helical molecules. “Oligonucleotide” generally refers topolynucleotides of between about 5 and about 100 nucleotides of single-or double-stranded DNA. However, for the purposes of this disclosure,there is no upper limit to the length of an oligonucleotide.Oligonucleotides are also known as oligomers or oligos and may beisolated from genes, or chemically synthesized by methods known in theart.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially pure” refers to a compound thatis removed from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

The term “transgene” is used herein to describe genetic material thathas been or is about to be artificially inserted into the genome of acell, particularly a mammalian cell for implantation into a livinganimal. The transgene is used to transform a cell, meaning that apermanent or transient genetic change, preferably a permanent geneticchange, is induced in a cell following incorporation of exogenous DNA. Apermanent genetic change is generally achieved by introduction of theDNA into the genome of the cell. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs, and the like. Ofinterest are transgenic mammals, e.g. cows, pigs, goats, horses, etc.,and particularly rodents, e.g. rats, mice, etc.

The terms “treatment” “treating” and the like are used herein toencompass any treatment of any disease or condition in a mammal,particularly a human, and includes: a) preventing a disease, condition,or symptom of a disease or condition from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; b) inhibiting a disease, condition, or symptom of a diseaseor condition, e.g., arresting its development and/or delaying its onsetor manifestation in the patient; and/or c) relieving a disease,condition, or symptom of a disease or condition, e.g., causingregression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. Other subjects mayinclude cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses,and so on.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acarboxyl-terminal truncated apoE protein” includes a plurality of suchproteins and reference to “the cell” includes reference to one or morecells and equivalents thereof known to those skilled in the art, and soforth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

ApoE Polypeptide Fragments

The present invention provides isolated apoE polypeptide fragments, andcompositions including such polypeptide fragments. ApoE polypeptidefragments include carboxyl-terminal truncated apoE; and fragments thatinclude at least amino acids 244-260 of apoE. Carboxyl-truncated apoEpolypeptides are useful in screening assays to identify agents thatreduce formation of neurofibrillary tangles, as described below.Fragments that include at least amino acids 244-260 of apoE are usefulto inhibit apoE binding to p-tau and p-NF-H, thereby reducing formationof neurofibrillary tangles.

Carboxyl-terminal truncations of apoE include any carboxyl-terminaltruncated form of apoE that binds both p-tau and p-NF-H and inducesformation of neurofibrillary tangles that include carboxyl-terminaltruncated apoE, p-tau, and p-NF-H in a neuronal cell. Whether a givencarboxyl-terminal truncated apoE polypeptide binds p-tau, binds p-NF-H,and forms neurofibrillary tangles in a neuronal cell is readilydetermined by those skilled in the art using known assays. For example,formation of complexes that include carboxyl-terminal truncated apoEpolypeptide, p-tau, and p-NF-H can be detected using antibodies specificfor each of these proteins, using, e.g., established techniques of cellstaining; established techniques for co-immunoprecipitation; protein(“Western”) blotting; and the like, e.g., as described in the Examples.Formation of neurofibrillary tangles can be assessed visually, e.g., byobserving intracellular inclusions in neuronal cells. Whether suchneurofibrillary tangles include carboxyl-terminal truncated apoE, p-tau,and p-NF-H can be determined using antibodies specific for each protein.

Deletion of from about 28 to about 30, from about 30 to about 35, fromabout 35 to about 40, from about 40 to about 45, or from about 45 toabout 48 amino acids from the carboxyl terminus of apoE3 or apoE4results in carboxyl-terminal truncated apoE that bind p-tau, bindp-NF-H, and induce formation of neurofibrillary tangles in a neuronalcell. Specific carboxyl-terminal truncated apoE polypeptides that giverise to neurofibrillary tangles include, but are not limited to,apoE4Δ272-299; apoE3Δ272-299; apoE4Δ261-299; and apoE4Δ252-299.

The invention further provides fragments of apoE that correspond to theportion of apoE that interacts with p-tau and p-NF-H. Such fragmentsinclude at least amino acids 244-260 of the apoE polypeptide(apoE244-260). Also included are apoE244-260 fragments that includeadditional carboxyl- and/or amino-terminus additions of from 1 to about5, from about 5 to about 10, from about 10 to about 15, or from about 15to about 20 amino acids. Such fragments are useful to inhibit binding ofapoE with p-tau and p-NF-H.

Nucleic Acids and Host Cells

The present invention provides nucleic acids that include a nucleotidesequence that encodes a carboxyl-terminal truncated apoE polypeptide, aswell as host cells that contain the nucleic acid. In some embodiments,the host cells are isolated. In other embodiments, the host cells arepart of a transgenic, non-human animal that includes, as a transgene, anucleic acid of the invention.

Nucleic Acids

The subject nucleic acid molecules may be part of a vector (“construct”)for use in generating a transgenic, non-human animal of the invention,as described below, or for use in generating a recombinant host cellthat produces a carboxyl-terminal truncated apoE polypeptide. Inaddition, a nucleic acid molecule of the invention may encode all orpart of a carboxyl-terminal truncated apoE polypeptide of the invention,and as such is useful, as part of an expression vector, in producingcarboxyl-terminal truncated apoE polypeptide.

The sequence of the mouse apoE gene is found under Genbank accessionnumber D00466. Various primate apoE gene sequences are found underGenBank accession numbers AF200508, AF200507, AF200506, and AH009953(Hylobates lar, or gibbon); AH009952, AF200503, AF200504, and AF200505(Pongo pygmaeus, or orangutan); AH009951, AF200500, AG200501, andAF200502 (Gorilla gorilla); AH009950, AF200497, AF200498, AF200499 (Pantroglodytes, or chimpanzee). Any apoE-encoding sequence can be modifiedto encode a carboxyl-terminal truncated apoE polypeptide as describedabove.

In some embodiments, nucleic acids of the invention include the openreading frame encoding carboxyl-terminal truncated apoE polypeptide, oneor more introns, may further include adjacent 5′ and 3′ non-codingnucleotide sequences involved in the regulation of expression, and aregenerally up to about 10 kb in total length, but possibly longer. TheDNA sequences encoding all or part of the recombinant apoE are genomicDNA or a fragment thereof. The apoE gene encoding carboxyl-terminaltruncated apoE may be introduced into an appropriate vector forextrachromosomal maintenance or for integration into the host.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, except for thosenucleotides encoding carboxyl-terminal amino acids, as discussed above,including all of the introns that are normally present in a nativechromosome. It may further include the 3′ and 5′ untranslated regionsfound in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence.

The sequence of this 5′ region, and further 5′ upstream sequences and 3′downstream sequences, may be utilized for promoter elements, includingenhancer binding sites, that provide for expression in tissues whereapoE is expressed. The tissue specific expression is useful fordetermining the pattern of expression, and for providing promoters thatmimic the native pattern of expression. Naturally occurringpolymorphisms in the promoter region are useful for determining naturalvariations in expression, particularly those that may be associated withdisease. Alternatively, mutations may be introduced into the promoterregion to determine the effect of altering expression in experimentallydefined systems. Methods for the identification of specific DNA motifsinvolved in the binding of transcriptional factors are known in the art,e.g. sequence similarity to known binding motifs, gel retardationstudies, etc. For examples, see Blackwell et al Mol Med 1:194-205(1995); Mortlock et al. Genome Res. 6:327-33 (1996); and Joulin andRichard-Foy Eur J Biochem 232:620-626 (1995).

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of apoEexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans acting factors thatregulate or mediate expression. Such transcription or translationalcontrol regions may be operably linked to carboxyl-terminal truncatedapoE polypeptide-encoding apoE gene in order to promote expression ofcarboxyl-terminal truncated apoE or other proteins of interest incultured cells, or in embryonic, fetal or adult tissues, and for genetherapy.

In some embodiments, regulatory elements include regulatory elementsthat result in neuronal cell-specific expression of the operably linkedcarboxyl-terminal truncated apoE-encoding nucleic acid. Neuronalcell-specific regulatory elements (including promoters, enhancers, andthe like) are known to those skilled in the art. Examples of neuronalcell-specific regulatory elements include those from a neuron-specificenolase (NSE) gene (Hannas-Djebarra et al. (1997) Brain Res. Mol. BrainRes. 46:91-99); a PDGF gene; a Th1 gene (e.g., mouse Thy1.2 (Caroni etal. (1997) J. Neurosci. Methods 71:3-9); a neurofilament gene (e.g.,NF-L, NF-M, and NF-L); a glial filament acidic protein gene; a myelinbasic protein gene; a microtubule associated protein genes; asynaptophysin gene; a tyrosine hydroxylase gene; and the like.

In other embodiments, a nucleic acid molecule of the invention comprisesa cDNA comprising sequences that encode a carboxyl-terminal truncatedapoE protein of the invention. The nucleic acid compositions used in thesubject invention may encode all or a part of the carboxyl-terminaltruncated apoE polypeptides as appropriate. Fragments may be obtained ofthe DNA sequence by chemically synthesizing oligonucleotides inaccordance with conventional methods, by restriction enzyme digestion,by PCR amplification, etc. For the most part, DNA fragments will be ofat least 15 nucleotides (nt), usually at least 18 nt, more usually atleast about 50 nt. Such small DNA fragments are useful as primers forPCR, hybridization screening, etc. Larger DNA fragments, i.e. greaterthan 100 nt are useful for production of the encoded polypeptide. Foruse in amplification reactions, such as PCR, a pair of primers will beused.

In some embodiments, a nucleic acid molecule of the invention comprisesnucleotide sequences of a genomic apoE gene, modified as described abovesuch that the encoded apoE protein is a carboxyl-terminal truncated apoEpolypeptide. In other embodiments, a nucleic acid molecule of theinvention comprises the coding regions of a apoE gene, modified asdescribed above such that the encoded apoE protein is acarboxyl-terminal truncated apoE polypeptide (e.g., a cDNA moleculeencoding a carboxyl-terminal truncated apoE).

The invention further provides nucleic acid molecules that comprise anucleotide sequence that encodes a carboxyl-terminal truncated apoEprotein that binds p-tau, binds p-NF-H, and induces formation ofneurofibrillary tangles in a neuronal cell, wherein the nucleic acidmolecules hybridize under stringent hybridization conditions to one of amouse genomic apoE gene, modified such that the encoded apoE protein isa carboxyl-terminal truncated apoE; and the coding region of a mouseapoE gene, modified as described above such that the encoded apoEprotein is a carboxyl-terminal truncated apoE. Examples of stringentconditions are hybridization and washing at 50° C. or higher and in0.1×SSC (9 mM NaCl/0.9 mM sodium citrate).

The invention further provides nucleic acid molecules that comprise anucleotide sequence that encodes a carboxyl-terminal truncated apoEprotein that binds p-tau, binds p-NF-H, and induces formation ofneurofibrillary tangles in a neuronal cell, wherein the nucleic acidmolecules have at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or higher, nucleotidesequence identity with one of a mouse genomic apoE gene, modified suchthat the encoded apoE protein is a carboxyl-terminal truncated apoE; andthe coding region of a mouse apoE gene, modified as described above suchthat the encoded apoE protein is a carboxyl-terminal truncated apoEpolypeptide.

Subject nucleic acid molecules may comprise other, non-apoE nucleic acidmolecules (“heterologous nucleic acid molecules”) of any length. Forexample, the subject nucleic acid molecules may be flanked on the 5′and/or 3′ ends by heterologous nucleic acid molecules of from about 1 ntto about 10 nt, from about 10 nt to about 20 nt, from about 20 nt toabout 50 nt, from about 50 nt to about 100 nt, from about 100 nt toabout 250 nt, from about 250 nt to about 500 nt, or from about 500 nt toabout 1000 nt, or more in length. For example, when used as a probe todetect nucleic acid molecules capable of hybridizing with the subjectnucleic acids, the subject nucleic acid molecules may be flanked byheterologous sequences of any length. Heterologous sequence include, butare not limited to, sequences encoding a reporter protein, and the like.

The subject nucleic acid molecules may also be provided as part of avector, a wide variety of which are known in the art and need not beelaborated upon herein. Vectors include, but are not limited to,plasmids; cosmids; viral vectors; artificial chromosomes (YAC's, BAC's,etc.); mini-chromosomes; and the like. Vectors are amply described innumerous publications well known to those in the art, including, e.g.,Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds.,Wiley & Sons. Vectors may provide for expression of the subject nucleicacids, may provide for propagating the subject nucleic acids, or both.

The subject nucleic acid molecules are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the DNA will be obtained substantially free of other nucleicacid sequences that do not include a sequence or fragment thereof of thesubject genes, generally being at least about 50%, usually at leastabout 90% pure and are typically “recombinant”, i.e. flanked by one ormore nucleotides with which it is not normally associated on a naturallyoccurring chromosome.

The subject nucleic acid compositions find use in the preparation of allor a portion of the carboxyl-terminal truncated apoE polypeptides of theinvention, as described above. For expression, an expression cassettemay be employed. The expression vector will provide a transcriptionaland translational initiation region, which may be inducible orconstitutive, where the coding region is operably linked under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. These controlregions may be native to a gene encoding the subject peptides, or may bederived from exogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzyme orother protein marker, e.g. β-galactosidase, green fluorescent protein,luciferase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, or any of theabove-described fragment, and up to the complete open reading frame ofthe gene. After introduction of the DNA, the cells containing theconstruct may be selected by means of a selectable marker, the cellsexpanded and then used for expression.

Proteins and polypeptides may be expressed in prokaryotes or eukaryotesin accordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, insect cells incombination with baculovirus vectors, or cells of a higher organism suchas vertebrates, particularly mammals, e.g. COS 7 cells, Neuro-2A cells,may be used as the expression host cells. In some situations, it isdesirable to express the gene in eukaryotic cells, where the encodedprotein will benefit from native folding and post-translationalmodifications. Small peptides can also be synthesized in the laboratory.Polypeptides that are subsets of the complete sequences of the subjectproteins may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. A wide variety ofsuch systems are known to those skilled in the art.

Host Cells

Recombinant host cells comprising a subject nucleic acid molecule mayserve as a source of carboxyl-terminal truncated apoE protein of theinvention. They may also serve in drug screening assays to identifyagents that reduce binding of carboxyl-terminal truncated apoE4 to p-tauand p-NF-H in a neuronal cell.

In some embodiments, of particular interest are mammalian cells thatnormally produce apoE, and cells that normally take up apoE from theirenvironment. Examples of such cells include neuronal cells, microglialcells, and astrocytes. Immortalized neuronal cells, microglial cells,and astrocytes are also of interest.

Transgenic, Non-Human Animals

The present invention provides transgenic, non-human animals,particularly transgenic, non-human mammals that include, as a transgene,an exogenous nucleic acid that includes a coding region for acarboxyl-terminal truncated apoE polypeptide. The transgenic, non-humananimals of the invention are useful for screening agents that reducebinding of carboxyl-terminal truncated apoE4 to p-tau and p-NF-H in aneuronal cell, and which therefore reduce the formation ofneurofibrillary tangles. Such compounds are candidate agents useful fortreating Alzheimer's disease and associated pathologies.

In many embodiments, the carboxyl-terminal truncated apoE-encodingtransgene includes neuronal cell-specific regulatory elements such thatthe carboxyl-terminal truncated apoE is produced primarily in neuronalcells. However, the carboxyl-terminal truncated apoE-encoding transgenedoes not necessarily include neuronal cell-specific regulatory elements,as neurofibrillary tangles will form only in those cells that producep-tau and p-NF-H, i.e., neuronal cells.

Methods of generating transgenic, non-human animals, particularlytransgenic, non-human mammals, are known in the art. See, e.g., U.S.Pat. Nos. 6,268,545; 6,255,554; 6,222,094; and 6,204,43; “TransgenicAnimal Technology” C. A. Pinkert, ed. (1997) Acad. Press; “Gene KnockoutProtocols” M. J. Tymms, et al., eds. (2001) Humana Press; and “GeneTargeting: A Practical Approach” A. L. Joyner, ed. (2000) Oxford Univ.Press.

Transgenic animals comprise an exogenous nucleic acid sequence presentas an extrachromosomal element or stably integrated in all or a portionof its cells, especially in germ cells. Unless otherwise indicated, itwill be assumed that a transgenic animal comprises stable changes to thegermline sequence. During the initial construction of the animal,“chimeras” or “chimeric animals” are generated, in which only a subsetof cells have the altered genome. Chimeras are primarily used forbreeding purposes in order to generate the desired transgenic animal.Animals having a heterozygous alteration are generated by breeding ofchimeras. Male and female heterozygotes are typically bred to generatehomozygous animals.

In some embodiments, the endogenous apoE gene is knocked out (e.g.,rendered non-functional). In the present invention, transgenic knockoutshave a partial or complete loss of function in one or both alleles ofthe endogenous apoE gene.

Where the transgenic animal is a knock-out, the target gene expressionis undetectable or insignificant. A knock-out of an endogenous apoE genemeans that function of the endogenous apoE protein has beensubstantially decreased so that expression is not detectable or onlypresent at insignificant levels. This may be achieved by a variety ofmechanisms, including introduction of a disruption of the codingsequence, e.g. insertion of one or more stop codons, insertion of a DNAfragment, etc., deletion of coding sequence, substitution of stop codonsfor coding sequence, etc. In some cases the exogenous transgenesequences are ultimately deleted from the genome, leaving a net changeto the native sequence. Different approaches may be used to achieve the“knock-out.” See U.S. Pat. Nos. 5,464,764, 5,627,059 and related patentsand publications to Capecchi et al. A chromosomal deletion of all orpart of the native gene may be induced, including deletions of thenon-coding regions, particularly the promoter region, 3′ regulatorysequences, enhancers, or deletions of gene that activate expression ofapoE genes.

A functional knock-out may also be achieved by the introduction of ananti-sense construct that blocks expression of the native genes (forexample, see Li and Cohen Cell 85:319-329 (1996)). “Knock-outs” alsoinclude conditional knock-outs, for example where alteration of thetarget gene occurs upon exposure of the animal to a substance thatpromotes target gene alteration, introduction of an enzyme that promotesrecombination at the target gene site (e.g. Cre in the Cre-lox system),or other method for directing the target gene alteration postnatally.

The apoE gene transgene encodes carboxyl-terminal truncated apoE and isa genetically manipulated sequence as discussed above, includingdeletions in the coding region such that the encoded protein is acarboxyl-terminal truncated apoE protein. The introduced sequenceencodes a carboxyl-terminal truncated apoE polypeptide, and may furtherinclude additional coding sequences, including, e.g., nucleotidesencoding a reporter protein (e.g., β-galactosidase, luciferase, greenfluorescent protein, and the like). The transgene includes acarboxyl-terminal truncated apoE-encoding nucleotide sequence operablylinked to a promoter, which may be constitutive or inducible, and otherregulatory sequences required for expression in the host animal. By“operably linked” is meant that a DNA sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules, e.g. transcriptional activator proteins,are bound to the regulatory sequence(s).

DNA constructs for homologous recombination will comprise a nucleotidesequence encoding carboxyl-terminal truncated apoE, and will includeregions of homology to the target locus. DNA constructs for randomintegration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.Methods in Enzymology 185:527-537 (1990).

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. SeeU.S. Pat. Nos. 5,387,742, 4,736,866 and 5,565,186 for methods of makingtransgenic animals. After transformation, the cells are plated onto afeeder layer in an appropriate medium. Cells containing the constructmay be detected by employing a selective medium. After sufficient timefor colonies to grow, they are picked and analyzed for the occurrence ofhomologous recombination or integration of the construct. Those coloniesthat are positive may then be used for embryo manipulation andblastocyst injection. Blastocysts are obtained from 4 to 6 week oldsuperovulated females. The ES cells are trypsinized, and the modifiedcells are injected into the blastocoel of the blastocyst. Afterinjection, the blastocysts are returned to each uterine horn ofpseudopregnant females. Females are then allowed to go to term and theresulting litters screened for mutant cells having the construct. Byproviding for a different phenotype of the blastocyst and the ES cells,chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allergenicor congenic grafts or transplants, or in in vitro culture.

Compositions

The present invention further provides compositions, includingpharmaceutical compositions, comprising the agents, polypeptides,polynucleotides, recombinant vectors, and host cells of the invention.These compositions may include a buffer, which is selected according tothe desired use of the agent, polypeptide, polynucleotide, recombinantvector, or host cell, and may also include other substances appropriateto the intended use. Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use. In some instances, the composition cancomprise a pharmaceutically acceptable excipient, a variety of which areknown in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (1995)“Remington: The Science and Practice of Pharmacy”, 19th edition,Lippincott, Williams, & Wilkins.

Screening Assays

The present invention provides methods for identifying a compound thatreduce the formation neurofibrillary tangles in a neuronal cell. Agentsthat reduce the level of neurofibrillary tangles in a cell include thosethat reduce proteolytic cleavage of apoE; and those that reduceinteraction of carboxyl-terminal truncated apoE with p-tau and p-NF-H.Test agents that have an effect in an assay method of the invention arecandidates for treating disorders related to carboxyl-terminal truncatedapoE.

In some embodiments, the assays are in vitro cell-based screeningmethods for identifying compounds that reduce the formation ofcarboxyl-terminal truncated apoE and/or that block the interaction ofthe carboxyl-terminal truncated apoE with p-tau and/or p-NF-H in a cell.

In some embodiments, in vitro cell-based screening methods foridentifying compounds that reduce the formation of carboxyl-terminaltruncated apoE involve the use of host cells that produce full-lengthapoE that is subsequently proteolytically processed to carboxyl-terminaltruncated apoE. The methods generally involve contacting a cell thatincludes a carboxyl-truncated form of apoE in its cytosol with acandidate agent, and determining the effect of the candidate agent onthe level of carboxyl-truncated form of apoE and/or the level ofneurofibrillary tangles in the cell, compared to a control. A suitablecontrol lacks the test agent.

In other embodiments, in vitro cell-based screening methods foridentifying compounds that block the interaction of thecarboxyl-terminal truncated apoE with p-tau and/or p-NF-H generallyinvolve the use of subject host cells that include a nucleic acidencoding a C-terminal truncated form of apoE. The methods generallyinvolve contacting a cell that includes a carboxyl-truncated form ofapoE in its cytosol with a candidate agent, and determining the effectof the agent on binding of apoE with p-tau and p-NF-H and/or the levelof neurofibrillary tangles in the cell, compared to a control. Asuitable control lacks the test agent.

In other embodiments, a screening assay of the invention is an in vitrocell-free method. In vitro cell-free screening methods generally involvecontacting an enzyme that mediates proteolytic cleavage of apoE with (1)a substrate for the enzyme, the substrate providing for a detectablesignal when the enzyme is active; and (2) a test agent. A control sampleincludes the enzyme and the substrate, but no test agent. The enzyme maybe, but need not be, purified. The enzyme may be in a lysate of a cellthat produces carboxyl-terminal truncated apoE; may be partiallyisolated; or may be substantially isolated (e.g., about 90%, about 95%,or about 98% pure).

Compounds identified using an in vitro screening assay of the inventionare candidate compounds for treating disorders arising from the presencein a neuronal cell of C-terminal truncated forms of apoE, including AD.

In other embodiments, screening assays are in vivo screening assays. Insome embodiments, in vivo screening methods for identifying compoundsthat reduce the formation of carboxyl-terminal truncated apoE involvethe use of transgenic non-human animals that include as a transgene anucleic acid that encodes full-length apoE, and that produce, inneuronal cells, full-length apoE that is subsequently proteolyticallyprocessed to carboxyl-terminal truncated apoE. In other embodiments, invivo screening methods for identifying compounds that block theinteraction of the carboxyl-terminal truncated apoE with p-tau and/orp-NF-H generally involve the use of subject transgenic non-human animalsthat include as a transgene a nucleic acid that encodes acarboxyl-terminal truncated form of ApoE, and that produce, in neuronalcells, a carboxyl-terminal truncated apoE.

The methods generally involve contacting the transgenic, non-humananimal with a test agent and determining the effect, if any, of a testagent on the level of carboxyl-terminal truncated apoE and/or the levelof neurofibrillary tangles in a neuronal cell of the animal, compared toa control. A suitable control includes the transgenic animal notcontacted with the test agent. Compounds identified using an in vivoscreening assay of the invention are candidate compounds for treatingdisorders arising from the presence in a neuronal cell of C-terminaltruncated forms of apoE, including AD.

Candidate agents (also referred to herein as “test agents”) encompassnumerous chemical classes, although typically they are organicmolecules, e.g., small organic compounds having a molecular weight ofmore than 50 and less than about 2,500 daltons. Candidate agentsgenerally comprise functional groups necessary for structuralinteraction with proteins, e.g., van der Waals interactions and hydrogenbonding, and may include at least an amine, carbonyl, hydroxyl orcarboxyl group, and may include at least two of the functional chemicalgroups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including, but not limited to:peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligopeptides. Alternatively, libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts are available or readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs. Newpotential therapeutic agents may also be created using methods such asrational drug design or computer modeling.

Screening may be directed to known pharmacologically active compoundsand chemical analogs thereof, or to new agents with unknown propertiessuch as those created through rational drug design. Efficaciouscandidates can be identified by phenotype, i.e. an arrest or reversal ofparticular cognitive behaviors in comparison with wild-type animals anda transgenic non-human animal of the invention.

Agents that have an effect in an assay method of the invention may befurther tested for cytotoxicity, bioavailability, and the like, usingwell known assays. Agents that have an effect in an assay method of theinvention may be subjected to directed or random and/or directedchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs. Such structuralanalogs include those that increase bioavailability, and/or reducedcytotoxicity. Those skilled in the art can readily envision and generatea wide variety of structural analogs, and test them for desiredproperties such as increased bioavailability and/or reduced cytotoxicityand/or ability to cross the blood-brain barriers.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components is added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

In Vitro Cell-Based Screening Assays

In some embodiments, the invention provides methods for identifying acompound that reduces the formation of neurofibrillary tangles in acell, generally involving contacting a cell in vitro with a test agent,and determining the effect, if any, of the test agent on the formationof neurofibrillary tangles in the cell. In some embodiments, the methodsprovide for detecting the presence and/or amount of carboxyl-terminaltruncated apoE in the cells, where a reduction in the amount ofcarboxyl-terminal truncated apoE generally results in a reduction in theamount of neurofibrillary tangles. A test agent that reduces the amountof carboxyl-terminal truncated apoE is generally one that reduces theformation of neurofibrillary tangles in the cell.

In some embodiments, in vitro cell-based screening methods foridentifying compounds that reduce the formation of carboxyl-terminaltruncated apoE involve the use of host cells that produce full-lengthapoE that is subsequently proteolytically processed to carboxyl-terminaltruncated apoE. Such host cells are generated by introducing into asuitable cell a nucleic acid that encodes full-length apoE. The methodsgenerally involve contacting a cell that produces full-length apoE inits cytosol with a candidate agent, and determining the effect of thecandidate agent on the level of carboxyl-truncated form of apoE and/orthe level of neurofibrillary tangles in the cell, compared to a control.A suitable control lacks the test agent.

In other embodiments, in vitro cell-based screening methods foridentifying compounds that block the interaction of thecarboxyl-terminal truncated apoE with p-tau and/or p-NF-H generallyinvolve the use of subject host cells that include a nucleic acidencoding a C-terminal truncated form of apoE. Suitable cells include asubject host cell that includes a carboxyl-terminal truncatedapoE-encoding nucleic acid. The methods generally involve contacting acell that includes a carboxyl-truncated form of apoE in its cytosol witha candidate agent, and determining the effect of the agent on binding ofapoE with p-tau and p-NF-H and/or the level of neurofibrillary tanglesin the cell, compared to a control. A suitable control lacks the testagent.

The cell used in the screening method is one that includescarboxyl-terminal truncated apoE in the cytosol, and also produces p-tauand p-NF-H. In general, the cell is a neuronal cell, and in manyembodiments, the cell is a neuronal cell line. Neuronal cell lines arewell known in the art, and include, but are not limited to, neuro-2Acells; B103; PC12; NT2; and the like. In some embodiments, the cell is asubject host cell.

The cell used in the screening method includes carboxyl-terminaltruncated apoE in the cytosol. Carboxyl-truncated apoE can be producedby the cell, or can be provided exogenously.

In some embodiments, a nucleic acid that includes a nucleotide sequencethat encodes apoE, as described above, is introduced into the cell, suchthat the carboxyl-terminal truncated apoE-encoding nucleic acid istransiently or stably expressed in the cell.

In other embodiments, a nucleic acid that includes a nucleotide sequenceencoding full-length apoE is introduced into the cell, and thefull-length apoE polypeptide that is produced undergoes proteolyticcleavage in the cell to yield carboxyl-terminal truncated apoEpolypeptide in the cytosol.

In other embodiments, the cell is contacted with carboxyl-terminaltruncated apoE polypeptide (“exogenous carboxyl-terminal truncated apoEpolypeptide”). The cell takes up the exogenous carboxyl-terminaltruncated apoE polypeptide from the medium. To facilitate uptake ofexogenous carboxyl-terminal truncated apoE polypeptide,carboxyl-terminal truncated apoE polypeptide can be complexed with acompound that facilitates uptake into eukaryotic cells. Such compoundsinclude, but are not limited to, very low density lipoprotein (VLDL),e.g., β-VLDL; phospholipid/apoE complex; cationic lipids; polyethyleneglycol; polylactic-glycolic acid copolymer; dextran; and the like.

Whether the test agent has an effect on the formation of neurofibrillarytangles in the cell is determined using any known technique. Forexample, cells are stained with detectably-labeled antibodies specificfor p-tau, p-NF-H, or apoE, to visualize intracellular inclusions ofneurofibrillary tangles.

In some embodiments, cells are analyzed to determine whether the testagent has an effect on the level of carboxyl-terminal truncated apoE.Whether the test agent is effective in reducing the amount ofcarboxyl-terminal truncated apoE polypeptide in the cell is generallydetermined by analyzing cell lysates for the presence and/or amount ofcarboxyl-terminal truncated apoE.

In Vitro Cell-Free Assays

In other embodiments, a screening assay of the invention is an in vitrocell-free method. In vitro cell-free screening methods generally involvecontacting an enzyme that mediates proteolytic cleavage of apoE with (1)a substrate for the enzyme, the substrate providing for a detectablesignal when the enzyme is active; and (2) a test agent. A control sampleincludes the enzyme and the substrate, but no test agent.

The enzyme may be, but need not be, purified. The enzyme may be in alysate of a cell that produces carboxyl-terminal truncated apoE; may bepartially isolated; or may be substantially isolated (e.g., about 90%,about 95%, or about 98% pure.

Suitable substrates are those that provide a detectable signal when thesubstrate is acted on by the enzyme. A non-limiting example of such asubstrate is a peptide of the formula (P₃)_(n)P₂P₁-X, where P₄P₃P₂P₁ isa peptide and X is a moiety (a “tag group”) that is linked to thecarboxyl terminus of the peptide, and that provides a detectable signalwhen cleaved from the peptide upon action by the enzyme; P₁ is ahydrophobic residue such as leucine, phenylalanine, or methionine; P₂ isproline; P₃ is a hydrophobic amino acid such as alanine; and n≧2.Non-limiting examples of suitable peptides include Ala-Ala-Pro-Phe (SEQID NO:1); Ala-Ala-Pro-Met (SEQ ID NO:2); Ala-Ala-Pro-Leu (SEQ ID NO:3);Ala-Ala-Ala-Ala-Pro-Phe (SEQ ID NO:4); and the like.

X is a tag group capable of being detected by assays that detectradiolabelled, photochemical, colorimetric, chromogenic, fluorescent,fluorogenic, phosphorescent, electrochemical, chemiluminescent orbioluminescent tags.

The compounds contain tags at the C-terminal position of the peptide.The tags are any known to those of skill in the art for use in enzymeassays. Tags include, but are not limited to, tags that are capable ofbeing assayed, generally quantitatively, by radiolabels, byphotochemical, colorimetric, chromogenic, fluorescent, fluorogenic,phosphorescent, electrochemical, chemiluminescent or bioluminescent orimmunoassays. Exemplary tags are those detectable in colorimetric,chromogenic, fluorescent, fluorogenic, chemiluminescent orbioluminescent assays. Further exemplary tags are those the include atag group that can be a radioactively tagged group, or a fluorogenictag, a chromogenic tag or a chemiluminescent tag. All of theseindicators form either an amide linkage or an ester linkage the P₁ aminoacid such that these linkages are cleavable by the enzyme.

Chromogenic and fluorogenic labels and the use thereof are known in thisart (see, e.g., U.S. Pat. Nos. 4,448,715; 3,884,896; 3,886,136;4,016,042; 4,028,318; 4,119,620; 4,147,692; 4,155,916; 4,191,808;4,191,809 4,207,232; and 4,167,449 which contain lists of specificchromogenic or fluorogenic substrates for various proteolytic enzymes;colorimetric substances are shown in U.S. Pat. Nos. 4,217,269, 4,210,497and 4,221,706).

Fluorogenic or fluorescent tags suitable for use in the present methodsinclude, but are not limited to, dansyl, 4-methylcoumaryl-7-amino,4-trifluoromethylcoumaryl-7-amino, naphthylamino, 7-oxycoumaryl,5-amino-iso-phthalic acid di(lower alkyl, preferably methyl or ethyl)ester, coumaryl-7-amino tagged with radioactive halogen or ³H, ornaphthylamino tagged with radioactive halogen of ³H. Exemplaryfluorogenic tags include 4-methylcoumaryl-7-amino or4-trifluoromethylcoumaryl-7-amino. When the tag is a fluorogenic tag, itcan be 4-methyl coumaryl-7-amino, 4-trifluoromethyl coumaryl-7-amino,naphthylamino, 7-oxy-coumaryl, 5-amino isophthalic acid diethyl ester,dansyl, coumaryl-7-amino, 4-trifluoromethyl coumaryl-7-amino,2-methylanthranilic acid.

Colorimetric or chromogenic tags suitable for use in the present methodsinclude, but are not limited to, para-nitroanilino, para-nitrophenoxy,ortho-nitrophenoxy, ortho-carboxyphenoxy, nitrophenylamino,1-carboxy-2-nitrophen-5-ylamino, 1-sulfo-2-nitrophen-5-ylamino,naphthylamino, μ-naphthylamino, β-naphthylamino, nitronaphthylamino,5-nitro-α-naphthylamino, methoxynaphthylamino,4-methoxy-μ-naphthylamino, quinonylamino, quinon-5-ylamino,nitroquinonylamino, 8-nitroquinon-5-ylamino, 4-methylcoumaryl-7-amino,4-trifluoromethylcoumaryl-7-amino, and naphthylamino tagged withradioactive halogen.

When the tag is a chromogenic tag, it can be, for example,p-nitro-anilino, p-nitro-phenyloxy, nitrophenylamino, naphthylamino,nitronaphthylamino, methoxynaphthylamino, quinolylamino,nitroquinolylamino, 4-trifluoromethyl coumaryl-7-amino, ornaphthylamino.

Chemiluminescent tags suitable for use in the present methods include,but are not limited to, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione), iso-luminol(6-amino-2,3-dihydro-1,4-phthalazinedione) andN-(4-aminobutyl)-N-ethyl-iso-luminol(6-(N-(4-aminobutyl)-N-ethylamino)-2,3-dihyrophthalazine-1,4-dione).See, Simpson et al. (1979) Nature 279:646.

Radiolabelled tags suitable for use in the present methods include, butare not limited to, either ¹⁴C- or ³H-labelled anilino, benzylamino orlower alkoxy; or a halo label, in a hydroxyanilino, naphthylamino,hydroxybenzylamino or coumaryl-7-amino group.

Alternatively, the tag can be a reporter, such as chemiluminescent tagsuch as, amino-isoluminol, amino-luminol or other luminol derivative; ora bioluminescent tag, such as a luciferin, particularly a coelentrazine,or a luciferase, that upon cleavage is able to react with a suitableluciferase and luciferin, respectively. Also contemplated areimmunoreporters, in which a reporter-labeled antibody (or antigen, i.e.,ligand) binds to an antigen (receptor) on X; and biotin/avidin linkedreporters.

In Vivo Screening Assays

In some embodiments, the assay methods involve contacting a transgenic,non-human animal of the invention with a test agent, and determining theeffect, if any, of the agent on formation of neurofibrillary tangles, orany disorder associated with the presence of neurofibrillary tangles, inthe animal.

In some embodiments, in vivo screening methods for identifying compoundsthat reduce the formation of carboxyl-terminal truncated apoE involvethe use of transgenic non-human animals that include as a transgene anucleic acid that encodes full-length apoE, and that produce, inneuronal cells, full-length apoE that is subsequently proteolyticallyprocessed to carboxyl-terminal truncated apoE. Transgenic, non-humananimals that include as a transgene a nucleic acid that includes anucleotide sequence encoding full-length apoE are known in the art. See,e.g., U.S. Pat. No. 6,046,381; Raber et al. (1998) Proc. Natl. Acad.Sci. USA. 95:10914-10919; Muttini (1999) J. Neurosci. 19:4867-4880.

In other embodiments, in vivo screening methods for identifyingcompounds that block the interaction of the carboxyl-terminal truncatedapoE with p-tau and/or p-NF-H generally involve the use of subjecttransgenic non-human animals that include as a transgene a nucleic acidthat encodes a carboxyl-terminal truncated form of apoE, and thatproduce, in neuronal cells, a carboxyl-terminal truncated apoE.

The methods generally involve contacting the transgenic, non-humananimal with a test agent and determining the effect, if any, of a testagent on the level of carboxyl-terminal truncated apoE and/or the levelof neurofibrillary tangles in a neuronal cell of the animal, compared toa control. A suitable control includes the transgenic animal notcontacted with the test agent. Compounds identified using an in vivoscreening assay of the invention are candidate compounds for treatingdisorders arising from the presence in a neuronal cell of C-terminaltruncated forms of apoE, including AD.

The screen using the transgenic animals of the invention can employ anyphenomena associated learning impairment, dementia or cognitivedisorders that can be readily assessed in an animal model. The screeningcan include assessment of phenomena including, but not limited to: 1)analysis of molecular markers (e.g., levels of carboxyl-terminaltruncated apoE protein in brain tissue; and formation of neuriteplaques); 2) assessment behavioral symptoms associated with memory andlearning; 3) detection of neurodegeneration characterized by progressiveand irreversible deafferentation of the limbic system, associationneocortex, and basal forebrain (neurodegeneration can be measured by,for example, detection of synaptophysin expression in brain tissue)(see, e.g., Games et al. Nature 373:523-7 (1995)). These phenomena maybe assessed in the screening assays either singly or in any combination.

Generally, the screen will include control values (e.g., the level ofneurofibrillary tangle formation in the test animal in the absence oftest compound(s)). Test substances which are considered positive, i.e.,likely to be beneficial in the treatment of apoE-mediated disorders,will be those which have a substantial effect upon formation ofneurofibrillary tangles, and associated disorders (e.g., test agentsthat are able to rescue behavioral disorders caused by generation ofcarboxyl-terminal truncated apoE).

Methods for assessing these phenomena, and the effects expected of acandidate agent for treatment of apoE-associated disorders, are known inthe art. For example, methods for using transgenic animals in variousscreening assays for, for example, testing compounds for an effect onAD, are found in WO 9640896, published Dec. 19, 1996; WO 9640895,published Dec. 19, 1996; WO 9511994, published May 4, 1995 (describingmethods and compositions for in vivo monitoring of Aβ; each of which isincorporated herein by reference with respect to disclosure of methodsand compositions for such screening assays and techniques). Examples ofassessment of these phenomena are provided below, but are not meant tobe limiting.

Pathological Studies

After exposure to the candidate agent, the animals are sacrificed andanalyzed by immunohistology for either: 1) neurofibrillary tangles(NFTs) in the brain and/or 2) levels of carboxyl-terminal truncatedapoE. The brain tissue is fixed (e.g, in 4% paraformladehyde) andsectioned; the sections are stained with antibodies reactive withcarboxyl-terminal truncated apoE, and/or p-tau, and/or p-NF-H. Secondaryantibodies conjugated with fluorescein, rhodamine, horse radishperoxidase, or alkaline phosphatase are used to detect the primaryantibody. These experiments permit identification of neurofibrillarytangles and the regionalization of these NFTs to specific areas of thebrain.

Sections can also be stained with other diagnostic antibodiesrecognizing antigens such as Alz-50, A2B5, Aβ, neuron-specific enolase,and others that are characteristic of neurodegeneration. Staining withthioflavins and congo red can also be carried out to analyzeco-localization of Aβ deposits within the neuritic plaques and NFTs.

Analysis of Carboxyl-Terminal Truncated ApoE Production

Western Blot Analysis: Protein fractions can be isolated from tissuehomogenates and cell lysates and subjected to Western blot analysis asdescribed by Harlow et al., Antibodies. A laboratory manual, Cold SpringHarbor, N.Y., (1988); Brown et al., J. Neurochem 40:299-308 (1983);Tate-Ostroff et al., Proc Natl Acad Sci 86:745-749 (1989)); and Huang etal. (2001) Proc. Natl. Acad. Sci. USA 98:8838-8843. A brief descriptionis given below.

The protein fractions can be denatured in Laemmli sample buffer andelectrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Theproteins are then transferred to nitrocellulose filters byelectroblotting. The filters are blocked, incubated with primaryantibodies, and finally reacted with enzyme conjugated secondaryantibodies. Subsequent incubation with the appropriate substrate (whichsubstrate provides for a detectable signal) reveals the position ofcarboxyl-terminal truncated apoE proteins.

Behavioral Studies

Behavioral tests designed to assess learning and memory deficits can beemployed. An example of such as test is the Morris Water maze (MorrisLearn Motivat 12:239-260 (1981)). In this procedure, the animal isplaced in a circular pool filled with water, with an escape platformsubmerged just below the surface of the water. A visible marker isplaced on the platform so that the animal can find it by navigatingtoward a proximal visual cue. Alternatively, a more complex form of thetest in which there are no formal cues to mark the platform's locationwill be given to the animals. In this form, the animal must learn theplatform's location relative to distal visual cues. Alternatively, or inaddition, memory and learning deficits can be studied using a 3 runwaypanel for working memory impairment (attempts to pass through twoincorrect panels of the three panel-gates at four choice points) (Ohnoet al. Pharmacol Biochem Behav 57:257-261 (1997)).

Studies of Animal Models of Neuronal Damage

Rodent models of neuronal damage, for example neuronal damage caused bycerebral ischemia, may be examined to determine the role of apoE3 andapoE4 in the extent of neuronal damage caused by traumatic events aswell as their role in neuronal remodeling, repair and recovery from suchinsults. Rodent models of cerebral ischemia, both global ischemia andfocal ischemia, are useful for studying mechanisms controlling theoccurrence of cerebral ischemia and potential therapeutic strategies fortreatment of injury caused by ischemic events. Animal models of globalischemia, which is usually transient, have widely affected brain areasbut typically give rise to neuronal alterations in selectivelyvulnerable brain regions. Examples of such models include, but are notlimited to, the two vessel occlusion model of forebrain ischemia, thefour vessel occlusion model of forebrain ischemia, and ischemia modelsinvolving elevated cerebrospinal fluid pressure. See Ginsberg and Busto,Stroke, 20:1627-1642 (1989), which is herein incorporated by reference.Models of focal ischemia may mimic ischemic stroke injury, and typicallygive rise to localized brain infarction. Examples of models of focalischemia include, but are not limited to, middle cerebral arteryocclusion, photochemically induced focal cerebral thrombosis, blood clotembolization, microsphere embolization and the like. See McAuley,Cerebrovasc. Brain Metab. Review, 7:153-180 (1995) which is hereinincorporated by reference.

Methods of Inhibiting Formation of Neurofibrillary Tangles

The present invention provides methods for inhibiting formation ofneurofibrillary tangles in a neuronal cell. The methods generallyinvolve reducing the formation of carboxyl-terminal truncated form ofapoE in a neuronal cell.

A reduction in the level of neurofibrillary tangles in a neuronal cellcan be accomplished by: (1) reducing a level of carboxyl-terminaltruncated apoE polypeptide; (2) reducing proteolytic cleavage of apoEpolypeptide to a carboxyl-terminal truncated apoE polypeptide; (3)reducing interaction of a carboxyl-terminal truncated apoE polypeptidewith p-tau; (4) reducing interaction of a carboxyl-terminal truncatedapoE polypeptide with p-NF-H; (5) reducing a level and/or an proteolyticactivity of an enzyme(s) that cleaves apoE to generate carboxyl-terminaltruncated apoE.

In some embodiments, the methods involve introducing into an affectedcell a peptide that includes at least amino acids 244-260 of apoE, andthat inhibits the binding of carboxyl-terminal truncated apoE to p-tauand p-NF-H.

In other embodiments, the methods involve contacting a cell thatproduces carboxyl-terminal truncated apoE with an agent that inhibitsthe proteolytic activity of an enzyme(s) that cleaves apoE to generatecarboxyl-terminal truncated apoE. In some embodiments, the agent is aninhibitor of chymotrypsin-like serine proteases. An agent that inhibitsa chymotrypsin-like serine protease and that inhibits formation ofcarboxyl-terminal truncated apoE can be used in the methods of theinvention.

Agents that are inhibitors of chymotrypsin-like serine proteases includethose disclosed in U.S. Pat. Nos. 5,288,707; 6,127,340; and 6,281,206.Agents that inhibit a chymotrypsin-like serine protease include, but arenot limited to, X-(aa₄)_(m)-(aa₃)_(n)-(aa₂)-(aa₁)-Z, wherein aa₁, aa₂,and aa₃ represent natural or unnatural acid residues and (aa₄)_(m) oneor more optional amino acid residues linked to the amino group of aa₃.Alternatively any one or more amino acids (aa) groups may be analoguesof amino acid residues in which the α-hydrogen is replaced by asubstituent. X represents H or a substituent on the N-terminal aminogroup, Z is —COOH or a C-terminal extension group (carboxy replacementgroup), for example as known in the art. In some compounds, Z is aheteroatom acid group, e.g. —B(OH)₂, —P(OH)₂ or PO(OH), or a derivativethereof, for example a carboxylic acid ester, a dioxo-boronate[—B(Osubstituent)₂] or a phosphate [—PO(Osubstituent)₂] or BF₂.Exemplary heteroatom analogue groups are —B(OH)₂ and —P(O)(OM)₂S(O)₂OH.In some embodiments, the P2-P1 natural peptide linkage is replaced byanother linking moiety other than an N-substituted P2-P1 natural peptidelinkage.

In some embodiments, an agent that inhibits formation ofcarboxyl-terminal truncated apoE is a peptide inhibitor. A non-limitingexample of such an inhibitor is a peptide of the formula (P₃)_(n)P₂P₁,where P₄P₃P₂P₁ is a peptide; P₁ is a hydrophobic residue such asleucine, phenylalanine, or methionine; P₂ is proline; P₃ is ahydrophobic amino acid such as alanine; and n≧2. Non-limiting examplesof suitable peptides include Ala-Ala-Pro-Phe (SEQ ID NO:1);Ala-Ala-Pro-Met (SEQ ID NO:2); Ala-Ala-Pro-Leu (SEQ ID NO:3);Ala-Ala-Ala-Ala-Pro-Phe (SEQ ID NO:4); and the like.

Therapeutic Agents

The invention provides agents identified using the methods describedherein. Agents that reduce a level and/or an activity ofcarboxyl-terminal truncated apoE are used to treat apoE4-relateddisorders. An effective amount of the active agent is administered tothe host, where “effective amount” means a dosage sufficient to producea desired result. Generally, the desired result is at least a reductionin the level of carboxyl-terminal truncated apoE and/or a reduction inthe level of neurofibrillary tangles in a neuronal cell as compared to acontrol. Generally, an agent identified by a screening method of theinvention is formulated with one or more pharmaceutically acceptableexcipients, as described in more detail below.

The invention provides therapeutic agents that reduces formation ofcarboxyl-terminal truncated apoE in a neuronal cell. Therapeutic agentsinclude those that inhibit an enzyme that catalyzes the proteolyticdegradation of apoE in neuronal cells (an “apoE cleavage enzyme”) toproduce carboxyl-terminal truncated apoE fragments; and agents thatreduce activation of an apoE cleavage enzyme in neuronal cells withAβ₁₋₄₂ and/or that inhibits an interaction between an apoE cleavageenzyme and Aβ₁₋₄₂. The therapeutic agents are specific, e.g., theyinhibit an apoE cleavage enzyme that produces carboxyl-terminaltruncated apoE fragments that induce formation of neurofibrillarytangles in neuronal cells, but not other enzymes in neuronal cells;and/or they reduce activation of an apoE cleavage enzyme in neuronalcells with Aβ₁₋₄₂, but do not substantially affect activation of otherenzymes in neuronal cells; and/or they inhibit an interaction between anapoE cleavage enzyme and Aβ₁₋₄₂ in neuronal cells, but not otherprotein-protein interactions in neuronal cells. The therapeutic agentsare useful in methods of the invention for treating a disorderassociated with the presence in a neuronal cell of carboxyl-terminaltruncated apoE.

In some embodiments, an agent that inhibits formation ofcarboxyl-terminal truncated apoE is a peptide inhibitor. A non-limitingexample of such an inhibitor is a peptide of the formula (P₃)_(n)P₂P₁,where P₄P₃P₂P₁ is a peptide; P₁ is a hydrophobic residue such asleucine, phenylalanine, or methionine; P₂ is proline; P₃ is ahydrophobic amino acid such as alanine; and n≧2. Non-limiting examplesof suitable peptides include Ala-Ala-Pro-Phe (SEQ ID NO:1);Ala-Ala-Pro-Met (SEQ ID NO:2); Ala-Ala-Pro-Leu (SEQ ID NO:3);Ala-Ala-Ala-Ala-Pro-Phe (SEQ ID NO:4); and the like.

In many embodiments, the agent is formulated with a pharmaceuticallyacceptable excipient. A variety of formulations are well known to thoseskilled in the art and are described in more detail below.

The invention provides a pharmaceutical preparation that includes: aninhibitor of a chymotrypsin-like protease inhibitor; an agent selectedfrom the group consisting of an acetylcholinesterase inhibitor, anon-steroidal anti-inflammatory agent, a cyclooxygenase-2 inhibitor, anda monoamine oxidase inhibitor; and a pharmaceutically acceptableexcipient.

In some embodiments, the invention provides compositions comprising aninhibitor of an enzyme that catalyzes the formation of carboxyl-terminaltruncated apoE and at least one other therapeutic agent. Agents thatinhibit an apoE cleavage enzyme are described above. Other therapeuticagents that can be formulated together with an inhibitor of an enzymethat catalyzes the formation of carboxyl-terminal truncated apoEinclude, but are not limited to, agents that are used to treatindividuals with AD, including, but not limited to, acetylcholinesteraseinhibitors, including, but not limited to, Aricept® (donepezil), Exelon®(rivastigmine), metrifonate, and tacrine (Cognex™); non-steroidalanti-inflammatory agents, including, but not limited to, ibuprofen andindomethacin; cyclooxygenase-2 (Cox2) inhibitors such as Celebrex; andmonoamine oxidase inhibitors, such as Selegilene (Eldepryl or Deprenyl).Any known inhibitor of chymotrypsin-like serine proteases can beformulated together with another therapeutic agent used to treat AD.Dosages for each of the above agents are known in the art, and can beused in a pharmaceutical preparation with an apoE cleavage enzymeinhibitor. For example, Aricept® is generally administered at 50 mgorally per day for 6 weeks, and, if well tolerated by the individual, at10 mg per day thereafter.

Pharmaceutically acceptable excipients are known to those skilled in theart, and have been amply described in a variety of publications,including, for example, A. Gennaro (1995) “Remington: The Science andPractice of Pharmacy”, 19th edition, Lippincott, Williams, & Wilkins.

The invention further provides kits that include an agent that inhibitsan apoE cleavage enzyme in a neuronal cell. The agent may be formulatedwith a pharmaceutically acceptable excipient. The kit may furtherinclude an additional agent for treating AD, as described above (e.g.,an acetylcholinesterase inhibitor; a non-steroidal anti-inflammatoryagent; a Cox-2 inhibitor; a monoamine oxidase inhibitor), which may alsobe formulated with a pharmaceutically acceptable excipient. The kit mayfurther include a device for administering the formulation(s) to anindividual. Devices include, but are not limited to, needles, syringes,catheters, and the like. The kit may further include information for useof the components of the kit, including information regarding dosing,administration, and the like. The information may be provided in anyformat, including, but not limited to, printed information.

Methods of Treating ApoE-Related Disorders

The present invention provides methods of treating apoE-relateddisorders (e.g., a disorder associated with the presence ofcarboxyl-terminal truncated apoE in a neuronal cell of the individual;e.g., AD) in an individual. The methods generally involve administeringto an individual having an apoE-related disorder an effective amount ofa compound that reduces formation of carboxyl-terminal truncated form ofapoE in a neuronal cell in the individual and/or reduces formation ofneurofibrillary tangles and/or reduces binding of carboxyl-terminaltruncated apoE to p-tau and p-NF-H.

An “effective amount” of a compound is an amount that reduces a level ofcarboxyl-terminal truncated form of apoE in a neuronal cell in theindividual and/or reduces a level of neurofibrillary tangles and/orreduces binding of carboxyl-terminal truncated apoE to p-tau and p-NF-Hby at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80% or more, compared to a level of carboxyl-terminaltruncated form of apoE in a neuronal cell in the individual and/or alevel of neurofibrillary tangles and/or binding of carboxyl-terminaltruncated apoE to p-tau and p-NF-H in the absence of the compound.

In some embodiments, the invention provides a method of treatingAlzheimer's disease. In some embodiments, the method involves assayingfor the presence of carboxyl-terminal truncated apoE in a neuronal cell;and administering an inhibitor of an enzyme that catalyzes the formationof carboxyl-terminal truncated apoE in a neuronal cell. In otherembodiments, the method involves administering an inhibitor of achymotrypsin-like serine protease in an amount effective to inhibit anenzyme that catalyzes the formation of carboxyl-terminal truncated apoEin a neuronal cell, wherein the enzyme is inhibited and the level ofneurofibrillary tangles in a neuronal cell in the individual is reduced.

The present invention further provides a method of treating anindividual clinically diagnosed with a condition associated with thepresence of carboxyl-terminal truncated apoE in a neuronal cell. Themethods generally comprises analyzing a biological sample that includesa neuronal cell from an individual clinically diagnosed with anapoE-related disorder for the presence of carboxyl-terminal truncatedapoE in a neuronal cell. The presence of carboxyl-terminal truncatedapoE in a neuronal cell confirms the clinical diagnosis of a conditionassociated with apoE.

A treatment plan that is most effective for individuals clinicallydiagnosed as having a condition associated with apoE is then selected onthe basis of the detected carboxyl-terminal truncated apoE in a neuronalcell. Treatment may include administering a composition that includes anagent that inhibits an apoE cleavage enzyme in a neuronal cell.

Information obtained as described above can be used to predict theresponse of the individual to a particular agent. Thus, the inventionfurther provides a method for predicting a patient's likelihood torespond to a drug treatment for a condition associated with apoE,comprising determining whether carboxyl-terminal truncated apoE ispresent in a neuronal cell, wherein the presence of a carboxyl-terminaltruncated apoE is predictive of the patient's likelihood to respond to adrug treatment for the condition.

Formulations, Dosages, and Routes of Administration

The invention provides formulations, including pharmaceuticalformulations, comprising an agent that reduces a level and/or anactivity of carboxyl-terminal truncated apoE. In general, a formulationcomprises an effective amount of an agent that reduces a level and/or anactivity of carboxyl-terminal truncated apoE. An “effective amount”means a dosage sufficient to produce a desired result, e.g., reductionin a level and/or an activity of carboxyl-terminal truncated apoE, areduction in neurofibrillary tangles, an improvement in learning,memory, etc. Generally, the desired result is at least a reduction in alevel and/or an activity of carboxyl-terminal truncated apoE as comparedto a control. An agent that reduces a level and/or an activity ofcarboxyl-terminal truncated apoE may delivered in such a manner as toavoid the blood-brain barrier, as described in more detail below. Anagent that reduces a level and/or an activity of carboxyl-terminaltruncated apoE may be formulated and/or modified to enable the agent tocross the blood-brain barrier, as described in more detail below.

Formulations

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredreduction in of a level and/or an activity of carboxyl-terminaltruncated apoE, reduction in any apoE4-associated neurological disorder,reduction in an apoE4-associated activity, etc.

Thus, the agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives-such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Other modes of administration will also find use with the subjectinvention. For instance, an agent of the invention can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A. R.Gennaro, (2000) Lippincott, Williams & Wilkins. The composition orformulation to be administered will, in any event, contain a quantity ofthe agent adequate to achieve the desired state in the subject beingtreated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of an agent that reduces formationof neurofibrillary tangles and/or reduces the level of carboxyl-terminaltruncated apoE and/or reduces interaction of apoE with p-tau and p-NF-Hcan be administered in a single dose. Alternatively, a target dosage ofan agent that reduces formation of neurofibrillary tangles and/orreduces the level of carboxyl-terminal truncated apoE and/or reducesinteraction of apoE with p-tau and p-NF-H can be considered to be aboutin the range of about 0.1-1000 μM, about 0.5-500 μM, about 1-100 μM, orabout 5-50 μM in a sample of host blood drawn within the first 24-48hours after administration of the agent.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

An agent that reduces formation of neurofibrillary tangles and/orreduces the level of carboxyl-terminal truncated apoE and/or reducesinteraction of apoE with p-tau and p-NF-H is administered to anindividual using any available method and route suitable for drugdelivery, including in vivo and ex vivo methods, as well as systemic andlocalized routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intratumoral,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as an apoE4-associatedneurological disorder and pain associated therewith. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the pathological condition, or at least the symptomsthat characterize the pathological condition.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject” and “patient”) are treatable accordingto the subject methods. Generally such hosts are “mammals” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In many embodiments,the hosts will be humans.

Kits with unit doses of the active agent, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Crossing the Blood-Brain Barrier

The blood-brain barrier limits the uptake of many therapeutic agentsinto the brain and spinal cord from the general circulation. Moleculeswhich cross the blood-brain barrier use two main mechanisms: freediffusion; and facilitated transport. Because of the presence of theblood-brain barrier, attaining beneficial concentrations of a giventherapeutic agent in the central nervous system (CNS) may require theuse of drug delivery strategies. Delivery of therapeutic agents to theCNS can be achieved by several methods.

One method relies on neurosurgical techniques. In the case of gravelyill patients such as accident victims or those suffering from variousforms of dementia, surgical intervention is warranted despite itsattendant risks. For instance, therapeutic agents can be delivered bydirect physical introduction into the CNS, such as intraventricular orintrathecal injection of drugs. Intraventricular injection may befacilitated by an intraventricular catheter, for example, attached to areservoir, such as an Ommaya reservoir. Methods of introduction may alsobe provided by rechargeable or biodegradable devices. Another approachis the disruption of the blood-brain barrier by substances whichincrease the permeability of the blood-brain barrier. Examples includeintra-arterial infusion of poorly diffusible agents such as mannitol,pharmaceuticals which increase cerebrovascular permeability such asetoposide, or vasoactive agents such as leukotrienes. Neuwelt andRappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J. Cereb.Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.11:638-643.

Further, it may be desirable to administer the pharmaceutical agentslocally to the area in need of treatment; this may be achieved by, forexample, local infusion during surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers.

Therapeutic compounds can also be delivered by using pharmacologicaltechniques including chemical modification or screening for an analogwhich will cross the blood-brain barrier. The compound may be modifiedto increase the hydrophobicity of the molecule, decrease net charge ormolecular weight of the molecule, or modify the molecule, so that itwill resemble one normally transported across the blood-brain barrier.Levin (1980) J. Med. Chem. 23:682-684; Pardridge (1991) in: Peptide DrugDelivery to the Brain; and Kostis et al. (1994) J. Clin. Pharmacol.34:989-996.

Encapsulation of the drug in a hydrophobic environment such as liposomesis also effective in delivering drugs to the CNS. For example WO91/04014 describes a liposomal delivery system in which the drug isencapsulated within liposomes to which molecules have been added thatare normally transported across the blood-brain barrier.

Another method of formulating the drug to pass through the blood-brainbarrier is to encapsulate the drug in a cyclodextrin. Any suitablecyclodextrin which passes through the blood-brain barrier may beemployed, including, but not limited to, J-cyclodextrin, K-cyclodextrinand derivatives thereof. See generally, U.S. Pat. Nos. 5,017,566,5,002,935 and 4,983,586. Such compositions may also include a glycerolderivative as described by U.S. Pat. No. 5,153,179.

Delivery may also be obtained by conjugation of a therapeutic agent to atransportable agent to yield a new chimeric transportable therapeuticagent. For example, vasoactive intestinal peptide analog (VIPa) exertedits vasoactive effects only after conjugation to a monoclonal antibody(Mab) to the specific carrier molecule transferrin receptor, whichfacilitated the uptake of the VIPa-Mab conjugate through the blood-brainbarrier. Pardridge (1991); and Bickel et al. (1993) Proc. Natl. AcadSci. USA 90:2618-2622. Several other specific transport systems havebeen identified, these include, but are not limited to, those fortransferring insulin, or insulin-like growth factors I and II. Othersuitable, non-specific carriers include, but are not limited to,pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.Certain prodrugs have been described whereby, upon entering the centralnervous system, the drug is cleaved from the carrier to release theactive drug. U.S. Pat. No. 5,017,566.

Subjects Suitable for Treatment with a Therapeutic Agent of theInvention

A variety of subjects are suitable for treatment with an agentidentified by a method of the invention. Suitable subjects include anyindividual, particularly a human, who has an apoE-associated disorder,who is at risk for developing an apoE-associated disorder, who has hadan apoE-associated disorder and is at risk for recurrence of theapoE-associated disorder, or who is recovering from an apoE-associateddisorder.

Such subjects include, but are not limited to, individuals who have beendiagnosed as having Alzheimer's disease; individuals who have sufferedone or more strokes; individuals who have suffered traumatic headinjury; individuals who have high serum cholesterol levels; individualswho have Aβ deposits in brain tissue; individuals who have had one ormore cardiac events; subjects undergoing cardiac surgery; and subjectswith multiple sclerosis.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Characterization of Carboxyl-Terminal Truncated apoE inNeuronal Cells

Methods and Materials

Minimum essential medium (MEM), N2-medium supplements, and fetal bovineserum (FBS) were purchased from Life Technologies, Inc.Paraformaldehyde, streptolysin-O (STP-O), and Triton-100 were from Sigma(St. Louis, Mo.). The enhanced chemiluminescence (ECL) chemiluminescencedetection kit for Western blots was from Amersham Life Science.Protein-A agarose was from Boehringer Mannheim. Recombinant human apoE3,apoE4, apoE3(Δ272-299), or apoE4(Δ272-299) were expressed in E. coli andpurified using established techniques.

Polyclonal anti-human apoE antibody was from Calbiochem.Anti-apoE-carboxyl-terminal antibody (amino acids 272-299) was obtainedby passing the polyclonal anit-apoE antibody for three times through aSepharose CL-4B column that was coupled with a mixture ofapoE3(Δ272-299) and apoE4(Δ272-299). The unbound fraction was used asanti-apoE-carboxyl-terminal antibody. Complete removal of antibodiesagainst apoE-amino terminal portion (amino acids 1-271) was confirmed bywestern blotting on full-length apoE and apoE(Δ272-299).

Phosphorylation-dependent monoclonal anti-tau antibodies, AT8. (p-Ser202and p-Thr205), AT100 (p-Ser212 and p-Thr214), AT180 (p-Thr231), AT270(p-Thr181) were from Endogen. Phosphorylation-independent monoclonalanti-tau antibody, tau-1 (de-p-Ser202 and de-p-Thr205) andphosphorylation-dependent monoclonal anti-NF-H antibody, RT97, were fromBoehringer Mannheim. Phosphorylation-dependent monoclonal anti-NF-Hantibody, SM-31, was from Stemberger Monoclonals Inc. Monoclonalanti-NF-68, NR4, anti-NF-160, NN18, anti-NF-200 (NF-H), N52, andmonoclonal anti-FLAG tag antibody, M2, were from Sigma. Fluoresceinisothiocyanate (FITC)-, Rodamine-, or Cy5-coupled anti-rabbit or mouseIgG were from Vector. HRP-coupled anti-rabbit or mouse IgG was fromDako.

Preparation of apoE expression vectors—Polymerase chain reaction(PCR)-based molecular cloning techniques were used to construct variousapoE expression vectors. PCR products encoding wild-type or modifiedforms of apoE3 or apoE4 were subcloned into a pFLAG-CMV-3 vector(Sigma), which has an N-terminal FLAG fusion peptide and a preprotrypsinsignal sequence, thus, leading to secretion of the expressed proteins,or a p-FLAG-CMV-4 vector (Sigma), which has an N-terminal FLAG fusionpeptide without a signal sequence, thus leading to expression ofproteins in the cytosol. For some experiments, PCR products encodingwild-type or modified forms of apoE3 or apoE4 were subcloned into apEGFP-C1 vector (Clontech), which has an amino-terminal greenfluorescent protein (GFP) fusion without a signal sequence, thus,leading to expression of GFP-apoE fusion proteins in the cytosol. Forall vectors, a human cytomegalovirus (CMV) immediate early promoterdrives expression of the proteins. All DNA constructs used in this studyand their properties were illustrated in Table I.

TABLE I DNA constructs used in this study and their propertiesConstructs Place of expression N-terminal fusion pFLAG-CMV-3 vectorApoE3 Secretory pathway FLAG ApoE4 Secretory pathway FLAGApoE3(Δ272-299) Secretory pathway FLAG ApoE4(Δ272-299) Secretory pathwayFLAG ApoE4(Δ261-299) Secretory pathway FLAG ApoE4(Δ252-299) Secretorypathway FLAG ApoE4(Δ244-299) Secretory pathway FLAG pFLAG-CMV-4 vectorApoE3 Cytosol FLAG ApoE3 Cytosol FLAG ApoE3(Δ272-299) Cytosol FLAGApoE4(Δ272-299) Cytosol FLAG ApoE4(Δ261-299) Cytosol FLAGApoE4(Δ252-299) Cytosol FLAG ApoE4(Δ244-299) Cytosol FLAG pEGFP-C1vector ApoE3 Cytosol GFP ApoE3 Cytosol GFP ApoE3(Δ272-299) Cytosol GFPApoE4(Δ272-299) Cytosol GFP ApoE4(Δ261-299) Cytosol GFP ApoE4(Δ252-299)Cytosol GFP ApoE4(Δ244-299) Cytosol GFP ApoE4(Δ1-232, Δ272-299) CytosolGFP ApoE4(Δ1-20, Δ272-299) Cytosol GFP ApoE4(Δ1-45, Δ272-299) CytosolGFP ApoE4(Δ1-84, Δ272-299) Cytosol GFP ApoE4(Δ1-126, Δ272-299) CytosolGFP ApoE4(Δ1-170, Δ272-299) Cytosol GFP

Cell cultures—Murine neuroblastoma (Neuro-2a) cells were obtained fromAmerican Type Culture Collection (Rockville, Md.). Neuro-2a cells weremaintained at 37° C. in a humidified 5% CO₂ incubator in MEM-containing10% FBS, supplemented with non-essential amino acids, penicillin, andstreptomycin.

Human teratocarcinoma cells (NT2) were maintained in Opti-MEM-I (GIBCO)containing 5% FBS and penicillin/streptomycin. The NT2 cells weredifferentiated into neuronal cells by retinoic acid treatment. Briefly,NT2 cells were incubated with 1 μM retinoic acid (RA) twice a week for 4weeks. The cells were then replated in DMEM high glucose (DMEM/HG)containing 10% FBS and penicillin/streptomycin. One day later, the NT2cells were replated on Matrigel-coated dishes (Collaborative Research).For the following 2 weeks, the differentiated NT2 (NT2-N) cells weremaintained in DMEM/HG containing 10% FBS, penicillin/streptomycin,1-β-D-arabinofuranosylcytosine (1 μM), fluorodeoxyuridine (10 μM), anduridine (10 μM). Thereafter, NT2-N cells were maintained in Opti-MEM-Icontaining 5% FBS and penicillin/streptomycin.

Primary cultures of cortical neurons were prepared from 17-day-old mouseembryos as previously described. Fiumelli et al. (1999) Eur. J.Neurosci. 11:1639-1646. Briefly, dissected cortexes were suspended in2.5% trypsin solution for 15 minutes at 37° C., washed three times withcalcium- and magnesium-free Hank's balanced salt solution, and thentriturated with a glass pipette to dissociate the cells. Cells were thenplated at a density of 2×10⁵/cm² on poly-lysine treated dishes orchamber slides in DME medium supplemented with 1 mM glutamine, 7.5 mMsodium bicarbonate, 5 mM HEPES buffer (pH 7.0), 0.1 mg/ml streptomycin,and 0.06 mg/ml penicillin. A mixture of hormones and salt containing 25μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine, 20 nMprogesterone, and 30 nM sodium selenate was added into the culturemedium. Cells were maintained for 6 days at 37° C. in a humidifiedatmosphere of 95% air and 5% CO₂. As reported previously (NS3307),immunostaining of 6 days in vitro cortical cultures with cell-specificantibodies yields >90% neuron-specific enolase-immunoreactive cells.

Transient transfection of cells—Neuro-2a, NT2-N, or cultured mouseprimary neurons were transiently transfected with various apoE cDNAconstructs, as listed in Table I, by using lipofectamine method.Briefly, 2 μg of apoE DNA were pre-incubated with 7 μl of lipofectaminein 200 μl of serum-free MEM containing N2-supplements at roomtemperature for 30 minutes. After addition of 800 μl of serum-free MEMcontaining N2-supplements, the mixture was added into the cells that hadbeen washed three times with serum-free MEM. Ten to forty-eight hourslater, the transfected cells were used for different experiments.

Immunocytochemistry—Neuro-2a cells were grown in 2-well chamber slidesfor 24 hours and were then transiently transfected with different apoE3or apoE4 DNA constructs, as described above. After washing twice in PBS,the transfected cells were fixed with 3% paraformaldehyde in PBS for 30minutes at room temperature, washed twice in PBS, and then permeabilizedwith Triton-X100 for 5 minutes which was followed by three washes inPBS.

To block nonspecific reactions, all slides were incubated for 1 hour atroom temperature with PBS containing 2% BSA and 10% normal serum(Vector) derived from the same species as the source of secondaryantibody (sheep or rabbit), followed by a 1-hour incubation with theprimary antibody (monospecific rabbit anti-human apoE, 1:500; monoclonalanti-p-tau, 1:100-1:1000, anti-p-NF-H, 1:100-1:500) in PBS containing 2%BSA.

Slides were then washed three times in PBS containing 2% BSA andincubated for 1 hour at room temperature with the secondary antibody(FITC-coupled anti-rabbit IgG to detect antigen-bond anti-human apoE,Rodamine- or Cy5-coupled anti-mouse IgG to detect antigen-bondanti-p-tau or anti-p-NF-H). After three washes in PBS containing 2% BSA,immunofluorescently labeled slides were mounted in VectaShield (Vector)and viewed with a MRC-1024 laser scanning confocal system (Bio-Rad)mounted on an Optiphot-2 microscope (Nikon, Tokyo, Japan).

For double labeling (apoE and p-tau, p-NF-H, or FLAG tag), the FITC andCy5 or Rodamine channels were viewed individually, and the resultingimages were pseudocolored in green (FITC) or red (Cy5 or Rodamine) withAdobe PhotoShop (version 5.5, Adobe System, San Jose, Calif.).

For the cells transiently transfected with GFP-apoE DNA constructs,after fixed in 3% paraformaldehyde and washed twice in PBS, the greenfluorescence was viewed directly with a MRC-1024 laser scanning confocalsystem (Bio-Rad) as described above. In some experiments, transientlytransfected Neuro-2a cells expressing GFP-apoE fragments wereimmunostained for p-tau and p-NF-H, as described above, to detect theco-localization of apoE fragments and p-tau and p-NF-H.

Differential permeabilization of cells with streptolysin-O and tritonx-100—In some experiments, to determine whether the intracellularinclusions is in the cytosol or in subcellular organelles, Neuro-2acells expressing apoE4 or apoE4(Δ272-299) were treated with seramin toremove cell surface-bond apoE, fixed with 3% paraformaldehyde, and thenincubated with either 500 U/ml of streptolysin-O (STP-O) for 15 minutesat 4° C., which permeabilizes the plasma membranes of the cells withoutrupturing the subcellular organelle membranes, or 0.5% triton X-100,which permeabilizes both plasma and subcellular membranes. Then,anti-apoE immunostaining was performed as described above.

Immunoprecipitation and western blotting—Neuro-2a cells were grown to80% confluence in 6-well plates or 15-cm dishes and were thentransiently transfected with different apoE DNA constructs, as describedabove. Twenty-four to forty-eight hours later, the cells were harvestedand lysed in ice-cold lysis buffer for 30 minutes. After spinning toremove nuclei (13,000 rpm for 15 minutes), apoE in supernatant wasimmunoprecipitated with monospecific rabbit anti-human apoE antibody andprotein-A agarose beads. The immunoprecipitates were washed twice withlysis buffer and three times with PBS. The presence of apoE, p-tau, orp-NF-H in the immunoprecipitates was analyzed by western blotting usingthe corresponding antibodies. Alternatively, the supernatants of celllysates were immunoprecipited with monoclonal anti-p-tau or anti-p-NF-Hantibodies and, then, the presence of apoE was analyzed by anti-apoEwestern blotting.

For some experiments, brain tissues from normal individuals orAlzheimer's disease patients were homogenized in ice-cold lysis buffer I(50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40, 0.5%sodium deoxycholate, and a mixtures of protease inhibitors). After spinat 14,000 rpm for 30 minutes at 4° C. to get the solublized proteins(supernatant), the pellet was further homogenized in ice-cold lysisbuffer II (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 4% SDS, 1% Nonidet P-40,1% sodium deoxycholate, and a mixtures of protease inhibitors). Bothsupernatant and the solubilized pellet were subjected to SDS-PAGE andanalyzed by western blotting as described above.

Electron microscopic analysis—Neuro-2a cells transiently transfectedwith DNA constructs encoding GFP or GFP-apoE4(Δ272-299) were lifted fromthe plates using 0.05% trypsin and 0.05 mM EDTA. The GFP positive cellswere sorted with fluorescence-activated cell sorter (FACS) and pelletedby centrifugation. The cells were then fixed for 1 hour with 2.5%glutaraldehyde in 0.1M cacodylate buffer and post-fixed for 1 hour in 2%OsO₄. The cells were dehydrated, embedded in Epon 812, sectioned (80 nm)using a Reichert Ultracut E, and stained with uranyl acetate and leadcitrate. The cells were then photographed using a JEOL CCX-100IIelectron microscope.

Results

Expression of ApoE4 Induces Formation of Intracellular Inclusions inNeuro-2a Cells.

To assess the role of apoE in NFT formation, the interaction of apoE3 orapoE4 with tau proteins in mouse neuroblastoma cells (Neuro-2a)transiently transfected with apoE3 or apoE4 cDNA constructs (Table I)was studied. Double-immunostaining of Neuro-2a cells expressing apoE4with a polyclonal anti-apoE antibody and a monoclonal anti-p-tauantibody revealed both apoE and p-tau immunoreactive intracellularinclusions in a subset of transfected cells. These intracellularinclusions were also recognized by a monoclonal antibody against p-NF-H.Phosphorylation of tau or NF-H within the intracellular inclusions wasconfirmed by alkaline phosphatase treatment, which abolished anti-p-tauor anti-p-NF-H immunostaining. These results suggest the presence of acomplex containing apoE, p-tau, and p-NF-H in a small number ofapoE4-transfected cells. However, immunostaining of Neuro-2a cellsexpressing apoE3 did not reveal the formation of intracellularinclusions.

Amylpoid-Beta (Aβ) and Endoplasmic Reticulum (ER) Stress Increase theFormation of Intracellular Inclusions in Neuro2a Cells Expressing ApoE4.

Since Aβ deposition is a major pathological component within the brainsof Alzheimer's disease patients, we determined the effect of Aβtreatment on the formation of apoE and p-tau immunoreactiveintracellular inclusions in Neuro2a cells expressing apoE. The resultsare shown in FIG. 1. Aβ1-42 treatment clearly increased the number ofinclusion-positive apoE4 expressing cells from 2.7±1.2% to 6.7±1.5%(p<0.001) and apoE3 expressing cells, from 0% to 2.8±0.6% (p<0.001).These results suggest that Aβ1-42 potentiates the formation ofintracellular inclusions in Neuro2a cells expressing apoE.

Since Aβ treatment of cells can cause ER stress (TN98), other ER stressinducers, including Brefeldin A (BfA) and tunicamycin (TN98), were usedto determine whether ER stress can increase the formation ofintracellular inclusions in apoE transfected Neuro2a cells. Incubationwith either BfA or tunicamycin increased the inclusion-positive cellsfrom 2.7±1.2% to 7.2±2.1% (p<0.001) in apoE4 expressing cells and from0% to 3.2±1.1% (p<0.001) in apoE3 expressing cells.

To test whether apoE directly interacts with p-tau and p-NF-H,coimmunoprecipitation studies were performed on cell lysates ofapoE4-transfected Neuro-2a cells treated with Aβ1-42 or BfA.Immunoprecipitation using anti-p-tau revealed the presence of 2-3 bands(molecular weight 50-60 Kd) detected by anti-p-tau antibody on westernblots. In addition, p-NF-H (molecular mass ˜200 Kd) was also detectedwithin the complex. However, anti-apoE western blotting did not revealfull-length apoE but rather a band with a lower molecular weight (˜30 Kdinstead of 34 Kd). These results confirm that the intracellularinclusions of the Neuro-2a cells are composed of p-tau and p-NF-H anddemonstrate that the apoE4 in the complex is a truncated form with amolecular weight 10-15% smaller than the full-length apoE.

Carboxyl-Terminal Truncated ApoE induces Formation of IntracellularInclusions in Neuro-2a Cells.

To determine whether the amino- or carboxyl-terminal truncation of apoEcan induce formation of the intracellular inclusions, Neuro-2a cellswere transfected with constructs possessing either amino- orcarboxyl-terminal truncations of apoE. Deletion of up to 28 (˜10%)amino-terminal amino acids of apoE4 did not result in formation of theintracellular inclusions. However, deletion of 28 (˜10%)carboxyl-terminal amino acids from either apoE3 (Δ272-299) or apoE4(Δ272-299) resulted in 32±5% or 78±8%, respectively, of the transfectedcells displaying the intracellular filamentous inclusions. Theintracellular inclusions in the Neuro-2a cells transfected withapoE4(Δ272-299) were immuno-reactive with anti-apoE, anti-p-tau, andanti-p-NF-H. There was complete merging of the immuno-reactivity of theinclusions, as shown for anti-apoE and anti-p-tau, indicatingco-localization of apoE and p-tau). The intracellular inclusions inducedby apoE3(Δ272-299) were similar but were smaller and were visible inmany fewer cells. These results suggest that carboxyl-terminal truncatedapoE, especially apoE4(Δ272-299), interacts with cytosolic p-tau andp-NF-H to induce the formation of intracellular inclusions in Neuro-2acells.

More precise intracellular localization of the inclusions wasascertained by differential permeabilization of the apoE4(Δ272-299)transfected Neuro-2a cells, followed by anti-apoE immunofluorescentstaining. Streptolysin-O (STP-O) treatment, which permeabilizes theplasma membrane without permeabilizing subcellular organelle membranes,resulted in no significant anti-apoE immunoreactivity in apoE4transfected Neuro-2a cells but strong anti-apoE immunoreactivity of theintracellular inclusions in apoE4(Δ272-299) transfected cells,suggesting the cytosolic distribution of the inclusions. However,Triton-X100 treatment, which permeabilizes both plasma and subcellularorganelle membranes, resulted in strong anti-apoE immunoreactivity inboth apoE4 and apoE4(Δ272-299) transfected cells. Taken together, theseresults indicate that the intracellular inclusions are cytosolic andsuggest that apoE4(Δ272-299) synthesized in the endoplasmic reticulum(ER) escapes the secretory pathway and enters the cytosol, leading tointeraction with cytosolic p-tau and p-NF-H. Alternatively, the secretedapoE4(Δ272-299) is re-internalized and then escapes theendosomal-lysosomal pathway and enters the cytosol. Support of the firstpossibility comes from the observation that the apoE4(Δ272-299)transfected cells secreted much less apoE into the medium than the apoE4cells, even thought they had similar apoE mRNA levels.

Cytosolic ApoE(Δ272-299) Induces Formation of Intracellular Inclusionsin Neuro-2a Cells.

We next tested whether direct expression of apoE(Δ272-299) in thecytosol of Neuro-2a cells would induce the formation of massiveintracellular inclusions. Neuro-2a cells were transfected withGFP-apoE3(Δ272-299) or GFP-apoE4(Δ272-299) DNA constructs lacking thesequence encoding the signal peptide. The green fluorescent protein(GFP) was fused to the N-terminus of the apoE to monitor theintracellular distribution. No apoE was secreted into the medium. Aftertransient transfection of Neuro-2a cells, GFP alone was distributed verydiffusely throughout the cells. Expression of GFP-apoE4(Δ272-299) in thecytosol, however, led to formation of massive intracellular filamentousinclusions in cell bodies, some of which extended into the neurites,whereas the GFP-apoE3(Δ272-299) had less ability to induce formation ofintracellular inclusions in Neuro-2a cells than the GFP-apoE4(Δ272-299).

In contrast, the GFP-ApoE3 (full-length) was distributed in thecytoplasm around the nuclei and in the neurites, with a punctate orvesicular distribution pattern. On the other hand, the distribution ofthe GFP-apoE4 (full-length) was less punctate than GFP-apoE3. Thepercentage of cells containing neurofibrillary tangle-like inclusionswas lower in GFP-apoE3(Δ272-299)-transfected cells, as compared to theGFP-apoE4(Δ272-299)-transfected cells, as shown in FIG. 2. These resultssuggest that the carboxyl-terminal 28 amino-acids of apoE protect apoEfrom interacting with p-tau and p-NF-H, thus preventing the formation ofthe intracellular inclusions.

Intracellular Inclusions Formed in Neuro-2a Cells ExpressingApoE4(Δ272-299) have Similar Ultrastructural and BiochemicalCharacteristics of Neurofibrillary Tangles Seen in Human Alzheimer'sDisease Brains.

The ultrastructure and composition of the intracellular inclusionsformed in Neuro-2a cells expressing cytosolic GFP-apoE4(Δ272-299) weredetermined. For ultrastructural analysis, electron microscopy revealedfilamentous aggregates formed in the Neuro-2a cells expressingGFP-apoE4(Δ272-299), but not in the cells expressing GFP alone.Filaments were randomly oriented, and were not membrane-bound. A highpower magnification revealed that the inclusion is composed of manystraight filaments, with a diameter of 10-20 nm, which are similar tosome tangles seen in human Alzheimer's disease brains. The cellsexpressing GFP-apoE4(Δ272-299) also showed many electron densemembrane-bound organelles, suggestive of degenerating organelles.

To characterize the composition of the intracellular inclusions in moredetail, two sets of monoclonal antibodies were used (Table II):anti-p-tau antibodies (AT8, AT100, AT180, AT270, and tau-2), whichrecognize phosphorylated serine or threonine residues at differentpositions in tau protein, and tau-1, which recognizes nonphosphorylatedtau protein; anti-neurofilament antibodies, RT97 and N14, whichrecognize the phosphorylated NF-H, and N52, which recognizesnonphosphorylated NF-H.

TABLE II Immunoreactivity of the NFT-like structures with variousmonoclonal antibodies against p-tau or p-NF-H Recognized residuesReactivity with NFT- Antibodies in tau or NF-H* like structures AT8p-Ser202, p-Thr205 + AT100 p-Ser212, p-Thr214 ++ AT180 p-Ser231 − AT270p-Thr181 +++ RT97 KSP motif +++ SM31 unknown ++ *the residues or motifin human tau or NF-H.

It has been reported that the tangles in brains of Alzheimer's diseasepatients contain both p-tau and p-NF-H. Immunofluorescent staining ofNeuro-2a cells expressing GFP-apoE4(Δ272-299) demonstrated that theinclusions were positive for some, but not all, of the monoclonalantibodies that recognize p-tau in paired helical filaments (PHF) inhuman Alzheimer's disease brains (Table II), suggesting that some, butnot all, serine or threonine residues of tau protein were phosphorylatedin the intracellular inclusions in Neuro-2a cells. Immunostaining withanti-p-NF-H antibody, RT97, demonstrated that the intracellularinclusion also contained p-NF-H.

To confirm the presence of both p-tau and p-NF-H, cell lysatescontaining the inclusions from apoE4(Δ272-299) transfected Neuro-2 cellswere immunoprecipitated with anti-apoE antibody. Then, anti-p-tauantibody western blotting revealed proteins with molecular mass of 40-60Kd, which were p-tau proteins. Likewise, anti-p-NF-H antibody (RT97)western blotting revealed p-NF-H with molecular mass of 200 Kd. Otherbands with molecular mass of 40-60 Kd, characteristic of tau isoforms,were also recognized by monoclonal antibody RT97. It has been reportedthat monoclonal antibody RT97 cross-reacts with phosphorylated tau;therefore, these bands are likely p-tau proteins.

On the other hand, if the cellular lysates from apoE4(Δ272-299)transfected cells were immunoprecipited with monoclonal antibodiesagainst p-tau or p-NF-H, anti-apoE western blotting revealed a band withmolecular mass of ˜30 KD, i.e., apoE4(Δ272-299). Taken together, theseresults suggest that apoE4(Δ272-299) forms a complex with p-tau andp-NF-H to form the intracellular inclusions in Neuro-2a cells. Theseintracellular inclusions have similar ultrastructural and biochemicalcharacteristics of neurofibrillary tangles (NFT) seen in humanAlzheimer's disease brains and, thus, we refer them to NFT-likestructures.

Amino Acids 244-260 of ApoE Interact with p-tau and p-NF-H to Form theNFT-Like Structures in Neuro-2a Cells.

To determine the region of apoE that interacted directly with p-tau andp-NF-H to form the NFT-like structures, an additional series ofcarboxyl-terminal truncations on the GFP-apoE4(Δ272-299) DNA constructs(without signal peptide) were prepared and transfected into Neuro-2acells. Sites of truncation are illustrated in FIG. 3A. The results areshown in FIG. 3B. Truncation to amino acid 260 (apoE4(Δ261-299)) did notalter the ability of the carboxyl-terminal truncated apoE4 to induceformation of the NFT-like structures, although truncation to amino acid252 (apoE4(Δ252-299)(slightly decreased the formation of theintracellular NFT-like structures. However, truncation to amino acid 244(apoE4(Δ244-299)) abolished the formation of the NFT-like structures,suggesting that there are critical residues in the region 244-260 thatare responsible for binding to p-tau and p-NF-H to form the NFT-likestructures in Neuro-2a cells.

To confirm this observation, a GFP-apoE(Δ1-232, Δ272-299) DNA constructencoding only 38 amino acids of apoE (amino acids 233-271) (withoutsignal peptide) was transfected into Neuro-2a cells. The results areshown in FIG. 3B. The apoE(Δ1-232, Δ272-299) induced formation ofNFT-like structures in Neuro-2a cells, although these structures weremuch smaller and occurred in somewhat fewer transfected cells ascompared with GFP-apoE4(Δ272-299) transfected cells. Thus, these resultssupport the conclusion that the apoE amino acids 244-260 are required toinduce formation of NFT-like structures in Neuro-2a cells and theamino-terminal domain of apoE influences the extent of NFT-likestructure formation.

Amino-Terminal Domain of ApoE Modifies the Ability of theCarboxyl-Terminal Truncated ApoE to Form the NFT-Like Structures.

Since the sequence of amino acids 244-260 is identical in apoE3 andapoE4, this raises the question as to why apoE4(Δ272-299) has a greaterability than apoE3(Δ272-299) to form the NFT-like structures. Todetermine whether the amino-terminal domain of apoE can modify theability of apoE amino acids 244-260 to induce formation of the NFT-likestructures, a series of amino-terminal truncations on theGFP-apoE4(Δ272-299) DNA construct were made, as shown in FIG. 3A. Theresults are shown in FIG. 3C. Truncation of the first 20 amino acids(Δ1-20, Δ272-299), the first α-helix (Δ1-45, Δ272-299), and the secondα-helix (Δ1-84, Δ272-299) did not alter the ability ofGFP-apoE4(Δ272-299) to induce formation of the NFT-like structures inNeuro-2a cells. However, removal of the third α-helix (Δ1-126, Δ272-299)decreased dramatically the ability of the GFP-apoE4(Δ272-299) to induceformation of the NFT-like structures. Truncation of the fourth α-helix(Δ1-170, Δ272-299) did not decrease further the formation of theNFT-like structures. These results suggest that the sequence in thethird α-helix (amino acids 85-126), which includes the position 112where cysteine occurs in apoE3 and arginine in apoE4, modifies theability of apoE4(Δ272-299) to induce formation of the NFT-likestructures.

Formation of the NFT-Like Structures Induced by ApoE4(4272-299) isSpecific for Neurons.

To determine whether or not the formation of the NFT-like structuresinduced by apoE4(Δ272-299) is specific for neurons, we transfected twoother neuronal cells, primary cultured embryonic mouse brain neurons anddifferentiated human teratocarcinoma NT2 cells (NT2-N), and fournon-neuronal cell lines with GFP-apoE4(Δ272-299) (without signalpeptide) (Table III).

TABLE III Neuron-specific formation of NFT-like structures induced byGFP-apoE4(Δ272-299) NFT-like Cell lines Cell type structures Neuronalcells Neuro-2a Neuron + NT2-N Neuron + Mouse primary neurons Neuron +Non-neuronal cells CHO Fibroblast − COS-7 Fibroblast − C-6 Astrocyte −HepG2 Hepatocyte − McA-RH7777 Hepatocyte − NFT-like structures weredetermined by anti-apoE and anti-p-tau double-immunofluorescent stainingas described in Experimental Procedures.

Expression of GFP-apoE4(Δ272-299) in the cytosol induced formation ofthe NFT-like structures in neuronal cells but not in non-neuronal cells,as shown in Table III, above. These results indicate that formation ofthe NFT-like structures induced by apoE4(Δ272-299) is specific forneurons, which is consistent with neuron-specific expression of tau andNF-H.

Internalized Exogenous ApoE4(Δ272-299) can Induce Formation of theNFT-Like Structures in Neuro-2a Cells.

In human brains, apoE is synthesized and secreted primarily byastrocytes, although human neurons are capable of producing apoE. Thesecreted apoE can be internalized by neurons. To determine whetherinternalized apoE4(Δ272-299) can induce formation of NFT-like structuresin neurons. Neuro-2a cells were incubated with recombinantapoE4(A272-299), which had been complexed with the lipid transportvehicle β-VLDL. After 30 hours incubation, neurite outgrowth and theformation of NFT-like structures in the cells were determined. Neuro-2acells incubated with lipid transport vehicle alone for 30 hours showedneurite outgrowth. However, cells incubated with exogenousapoE4(Δ272-299) changed the morphology of the cells and did not showneurite outgrowth, suggesting that internalized apoE4(Δ272-299) mightimpair cytoskeletal structure and function. Anti-apoE immunofluorescentstaining demonstrated that the NFT-like structures were formed inNeuro-2a cells incubated with exogenous apoE4(Δ272-299). Thesestructures were identical to the tangle-like structures formed inNeuro-2a cells expressing GFP-apoE4(Δ272-299) in the cytosol. Thereforeinternalized apoE4(Δ272-299) can escape the endosomal-lysosomal pathwayand interact with cytosolic p-tau and p-NF-H.

Carboxyl-Terminal Truncated ApoE can be Generated Inside Neurons.

To determine whether the carboxyl-terminal truncated forms of apoEgenerated naturally inside neurons, Neuro-2a cells were transfected withfull-length apoE3 or apoE4 (with signal peptide), or Neuro-2a cells wereincubated with exogenous full-length apoE3 or apoE4. After lysis of thecells, intracellular apoE was immunoprecipitated with a monospecificanti-apoE antibody. Anti-apoE western blotting revealed a full-lengthapoE band and a lower molecular weight band, the molecular mass of whichwas similar to apoE(Δ272-299) (˜30 Kd). More fragments were generatedfrom apoE4 than from apoE3. The lower molecular weight band represents acarboxyl-terminal truncated form of apoE, as detected by a monoclonalantibody, 6C5, which recognizes the amino-terminal 15 amino acids.Therefore, the shorter fragment is the carboxyl-terminal truncated formof apoE composed of approximately 270 amino acids. This conclusion wasconfirmed in transfected Neuro-2a cells expressing apoE4 with anamino-terminal FLAG tag. Anti-FLAG antibody also recognized a proteinband with molecular mass of ˜30 Kd.

Since Aβ1-42 increased formation of intracellular inclusions in Neuro2acells expressing apoE4, we next determined whether treatment of thetransfected cells with Aβ will increase generation of thecarboxyl-terminal truncated form of apoE. Neuro-2a cells expressingapoE3 or apoE4 were incubated with Aβ1-42, then, intracellular apoE wasimmunoprecipitated and detected by anti-apoE western blotting. Treatmentof the transfected cells with Aβ1-42 significantly increased the amountsof the carboxyl-terminal truncated forms of both apoE3 and apoE4, butmore so with apoE4. Similar results were also obtained when exogenousapoE3 or apoE4 were incubated with Neuro-2a cells in the presence ofAβ1-42. These results suggest that Aβ1-42 may activate some unknownproteolytic enzymes that cleave apoE, especially apoE4, at itscarboxyl-terminus, creating a biologically active truncated form.

Carboxyl-Terminal Truncated Forms of apoE are Found in Brains ofAlzheimer's Disease Patients.

To determine whether carboxyl-terminal truncated forms of apoE can begenerated in vivo in human brains, we analyzed apoE in brain lysatesfrom normal or Alzheimer's disease patients by western blotting usingpolyclonal antibodies against full-length apoE or carboxyl-terminalportion of apoE (amino acids 272-299). The results are shown in FIGS.4A-E.

Polyclonal anti-apoE revealed full-length apoE in both the supernatantand the pellet of brain lysates from normal and Alzheimer's diseasepatients. An apoE fragment with molecular mass of ˜29 Kd was alsorecognized by polyclonal anti-apoE in supernatant from both normal andAlzheimer's disease brains, but to a greater extent in Alzheimer'sdisease brains. The 29-Kd apoE fragment was also found in the pellet ofAlzheimer's disease brains, but not in the pellet of normal brains. Inaddition, smaller apoE fragments with molecular mass of ˜14-19 Kd werefound in both supernatant and the pellet of Alzheimer's disease brainsbut not of normal brains.

However, western blotting using antibody against carboxyl-terminalportion of apoE (amino acids 272-299) only revealed full-length apoE butnot those apoE fragments in both supernatant and the pellets, suggestingthat the fragments are carboxyl-terminal truncated forms of apoE. Alsonote that large complexes (molecular mass over 220 Kd) were detected inthe pellets of Alzheimer's disease brains by anti-full-length apoE andanti-p-tau but not by anti-carboxyl-terminal apoE, suggesting that theselarge complexes contain carboxyl-terminal truncated forms of apoE andp-tau. Taken together, these results suggest that carboxyl-terminaltruncated forms of apoE are generated to a much greater extent inAlzheimer's disease brains than in normal brains, and most of them arepresent in insoluble forms, probably forming complexes with p-tau.

Example 2 Analysis of Transgenic Mice Expressing apoE3 or apoE4Specifically in CNS Neurons

Generation of Transgenic Mice

Transgenic mice expressing human apoE3 or apoE4 specifically in centralnervous system (CNS) neurons were generated at the Gladstone Institutesas reported previously (Raber J. et al. Proc. Natl. Acad. Sci. USA.1998, 95:10914-10919; Muttini M. et al. J. Neurosci. 1999,19:4867-4880). The expression of apoE transgenes was driven by aneuron-specific enolase (NSE) promoter. NSE-apoE3 and NSE-apoE4 lineswith matched cerebral levels of transgene expression were selected andcrossed with Apoe^(−/−) mice (C57BL/6J-Apoetm1Unc) from JacksonLaboratories (Bar Harbor, Me.). After elimination of wild-type mouseApoe alleles in two generations of breedings among the resultingoffspring, transgenic mice were further crossed with Apoe^(−/−) mice togenerate NSE-apoE3 and NSE-apoE4 mice that had at least 95% C57BL/6Jgenetic background. Crosses of NSE-apoE3 or NSE-apoE4 withC57BL/6J-Apoetm1Unc mice from Jackson Laboratories also yieldednontransgenic Apoe^(−/−) littermates, which were used as controls.

Genotyping of Transgenic Mice

Mice transgenic for NSE-apoE3 or NSE-apoE4 were identified by Southernblot analysis of genomic tail DNA using a DNA probe for human APOE.NSE-apoE3 and NSE-apoE4 mice were differentiated by PCR. Because thehuman APOE intron 3 was included in the NSE-apoE4 but not in theNSE-apoE3 construct, the amplicon generated with intron 3-spanningprimers (forward primer: nucleotides 3158-3175; reverse primer:nucleotides 3815-3834, GenBank accession number M10065) was 670 basepairs (bp) in NSE-apoE4 mice and 100 bp in NSE-apoE3 mice. ProteinaseK-digested tail tissue (1:100 dilution, 2 μl) was subjected to touchdownPCR in a total reaction volume of 25 μl with each primer (0.2 μM), dNTPs(dATP, dCTP, dGTP, dTTP, 200 μM each), and 0.15 μl of AmpliTaq GoldR DNApolymerase (Perkin-Elmer, Norwalk, Conn.). The reaction was run on aGeneAmp PCR System 9600 thermocycler (Perkin-Elmer). Polymerase chainreaction (PCR) products were analyzed on 1.5% agarose gels.

Age-Dependent Accumulation of C-Terminal Truncated Forms of ApoE4 inBrains of NSE-ApoE4 Transgenic Mice

Since only female NSE-apoE4 mice displays neurodegenerative phenotypesafter six months of age (Raber J. et al. Proc. Natl. Acad. Sci. USA.1998, 95:10914-10919; Muttini M. et al. J. Neurosci. 1999,19:4867-4880), we determined whether C-terminal truncated forms of apoE4could be found in these mice. Anti-full-length apoE immunoblot revealedmarked accumulation of the C-terminal truncated apoE fragments withmolecular weight of 14-20 kDa in the brain lysate of a 7 month oldNSE-apoE4 mouse, but not in the brain lysate of an age-matched NSE-apoE3mouse. There was an age-dependent accumulation of the C-terminaltruncated apoE4 fragments in both the supernatant and the solubilizedpellet fractions of NSE-apoE4 mouse brains. As shown in FIG. 5, muchless C-terminal truncated apoE3 fragments were found in the oldNSE-apoE3 mouse brains. These results indicate that, like in human ADbrains, the C-terminal truncated apoE4 fragments accumulated in brainsof NSE-apoE4 mice with an age-dependent manner.

Age-Dependent Accumulation of p-Tau in Brains of NSE-ApoE4 TransgenicMice

Anti-p-tau (AT8) immunoblot revealed accumulation of the soluble p-tau(˜50-60 kDa) in both supernatant and pellet fractions of brain lysatesof NSE-apoE4 mice, but not of NSE-apoE3 mice, with ages up to 9 monthold. The amount of the soluble p-tau (˜50-60 kDa) decreased in thesupernatant of brain lysates of NSE-apoE4 mice at the age of 18 months,whereas it increased in the supernatant of brain lysates of NSE-apoE4mice with a similar age. However, as shown in FIG. 6, the insolublep-tau (>200 kDa) dramatically accumulated in pellets of brain lysates ofNSE-apoE4 mice at the age of 18 months. The insoluble p-tau (>200 kDa)was also increased in pellets of brain lysates of NSE-apoE3 mice at theage of 18 months, but to a much less degree as compared with theage-matched NSE-apoE4 mice. Thus, there was an age-dependentaccumulation of both soluble and insoluble p-tau in brains of NSE-apoE4transgenic mice, which is similar to those observed in human AD brains.

Occurrence of p-Tau (AT8) Positive Intraneuronal Inclusions in theHippocampus of NSE-ApoE4 Transgenic Mice

Immunostaining of brain sections with monoclonal antibody AT8, whichrecognizes the phosphorylated Ser202 and Ser205 residues of tau in NFTs,revealed intraneuronal inclusions in the CA3 region and the hilus ofdentate gyrus in the hippocampus of NSE-apoE4 mice. These p-tau positiveintraneuronal inclusions contained also apoE, which is the C-terminaltruncated forms. As shown in FIG. 7, much less p-tau positiveintraneuronal inclusions were found in the hippocampus of NSE-apoE3mice. Therefore, the C-terminal truncated apoE may stimulate tauphosphorylation and induce p-tau positive intraneuronal inclusions inNSE-apoE4 transgenic mice.

Presence of an ApoE Cleavage Enzyme in Mouse Brain Lysates

Accumulation of the C-terminal truncated apoE4 in brains of NSE-apoE4transgenic mice suggests the presence of apoE cleavage enzyme(s) inmouse brains, which preferentially cuts apoE4. To test this hypothesis,purified human apoE3 or apoE4 were incubated with brain lysates of apoEknockout mice at 37° C. for 3 hours and the proteolysis of apoE wasanalyzed by anti-apoE western blot. Anti-apoE revealed full-length apoE,apoE fragments of 28-30 kDa, and the fragments of 14-20 kDa, with muchmore fragments being generated from apoE4 than from apoE3.Anti-C-terminal apoE western blot indicated that these fragments werethe C-terminal truncated forms of apoE. These results suggest that thereis an enzyme(s) in mouse brain that cleaves apoE to generate C-terminaltruncated fragments similar to those seen in human AD brains and inNSE-apoE4 transgenic mice. The results also suggest that apoE4 is muchmore susceptible to proteolytic cleavage.

Example 3 Characterization of an apoE Cleavage Enzyme

The apoE Cleavage Enzyme is a Serine Protease

To determine the biochemical properties of the putative apoE cleavageenzyme, we tested the effects of different known inhibitors for fourmajor categories of proteases, i.e., serine proteases, cysteineproteases, aspartate proteases, and metalloproteases. Both EDTA and EGTAdid not inhibit the cleavage of apoE4, suggesting that the enzyme is nota metalloprotease. Likewise, pepstain and Iodoacetimide or E-64 did notinhibit the cleavage of apoE4, indicating that the enzyme is not anaspartate protease nor a cysteine protease. PMSF totally abolished thecleavage activity, suggesting that the enzyme is a serine protease.Consistent with this conclusion, the complete inhibitor cocktail, whichcontains PMSF, also significantly inhibited the cleavage of apoE4.

The ApoE Cleavage Enzyme is a Chymotrypsin-Like Serine Protease.

Serine proteases can be categorized into the following groups:trypsin-like serine proteases, which cleave at the carboxyl side ofbasic residues (i.e., Arg or Lys), chymotrypsin-like serine proteases,which cleave at the carboxyl side of aromatic side chains (i.e., Phe orTrp) and of hydrophobic residues with larger side chain (i.e., Met), andelastase-like seine proteases, which cleave at the carboxyl side ofhydrophobic residues (i.e., Ala or Val). To test the specificity of theapoE cleavage enzyme, we incubated many tri- or tetra-peptides withArg/Lys, Phe, or Val/Ala at the C-terminal end, which are potentialsubstrates for trypsin-like, chymotrypsin-like, and elastase-like seineproteases, respectively, with roughly purified apoE cleavage enzyme.

To monitor the cleavage activity, a chromogenic group (p-nitroanilide)was added to the carboxyl-side of the last amino acid at the C-terminus.If the enzyme cuts the bond between the last amino acid andp-nitroanilide, the released p-nitroanilide will yield a yellow color,which can be monitored by a spectrophotometer at about 390-410 nm. FIG.8 shows representative results obtained from these peptide substrates.

The results demonstrate that the putative apoE cleavage enzyme is achymotrypsin-like serine protease, because it cuts at the carboxyl sideof aromatic side chains (phenylalanine, Phe) and of hydrophobic residueswith larger side chain (leucine, Leu; methionine, Met). Importantly, aproline at the P2 position and two hydrophobic amino acids (such asalanine, Ala) at the P3 and P4 positions are critical for determiningthe efficiency of the cleavage. Taken together, the putative apoEcleavage enzyme is a chymotrypsin-like serine protease, which recognizesthe motif of Ala-Ala-Pro-Phe (SEQ ID NO:1), Ala-Ala-Pro-Met (SEQ IDNO:2), Ala-Ala-Pro-Leu (SEQ ID NO:3), or a similar sequence in thesubstrate peptides or proteins. By using Ala-Ala-Pro-Phe-p-nitroanilideas a substrate, the chromogenic assay can be used as a high-throughputmethod to screen drugs, agents, or chemical compounds that inhibit apoEcleavage activity. Para-nitroanilide is detected spectrophotometricallyat about 390-410 nm.

Ala-Ala-Pro-Phe (SEQ ID NO:1), Ala-Ala-Ala-Ala-Pro-Phe (SEQ ID NO:4),and Ala-Ala-Pro-Leu (SEQ ID NO:3) are Peptide Inhibitors for the apoECleavage Enzyme.

By using the chromogenic assay, in which 1 mMAla-Ala-Pro-Phe-p-nitroanilide was used as the substrate, we screenedmany peptides towards the inhibition of the apoE cleavage enzymeactivity. As demonstrated in FIG. 9, 6 mM of peptides Ala-Ala-Pro-Phe(SEQ ID NO:1), Ala-Ala-Ala-Ala-Pro-Phe (SEQ ID NO:4), andAla-Ala-Pro-Leu (SEQ ID NO:3) inhibited apoE cleavage enzyme activity by67%, 58%, and 56%, respectively, at 20 hour incubation. Interestingly, 7μM apoE4 inhibited the apoE cleavage enzyme activity by 89%.

The peptides Ala-Ala-Pro-Phe (SEQ ID NO:1) and Ala-Ala-Pro-Leu (SEQ IDNO:3) were further tested for the inhibition of apoE cleavage. Afterincubation of a peptide (1 mM) with 4 μg of apoE4 for 3 hours at 37° C.in the presence of the partially purified apoE cleavage enzyme,anti-apoE western blot revealed significant inhibition of apoE cleavageby both peptides. Thus, both Ala-Ala-Pro-Phe (SEQ ID NO:1) andAla-Ala-Pro-Leu (SEQ ID NO:3) can be used as peptide inhibitors for theapoE cleavage enzyme.

It is evident from the above examples that the present inventionprovides carboxyl-terminal truncated apoE polypeptides; host cells thatinclude carboxyl-terminal truncated apoE; methods of using transgenicanimal models of carboxyl-terminal truncated apoE for identifyingcompounds that reduce formation of carboxyl-terminal truncated apoE; andcompounds that inhibit the formation of apoE. Carboxyl-truncated apoEpolypeptides are useful in generating host cells containing apoE for usein screening assays for compounds that inhibit the interaction ofcarboxyl-terminal truncated apoE with p-tau and p-NF-H. Compounds thatinhibit the interaction of carboxyl-terminal truncated apoE with p-tauand p-NF-H, and compounds that reduce formation of carboxyl-terminaltruncated apoE, reduce the formation of neurofibrillary tangles and aretherefore useful in treating disorders associated with the formation ofneurofibrillary tangles, including Alzheimer's disease, and relateddisorders.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of screening for biologically active agents that modulate aphenomenon associated with Alzheimer's disease (AD), the methodcomprising: (a) contacting a cell that produces a neurotoxic,hydrophobic, lipid-binding, carboxyl-terminal truncated apolipoprotein E(apoE) polypeptide with a test agent, wherein the neurotoxiccarboxyl-terminal truncated apoE polypeptide comprises amino acids244-260 of apoE; and (b) determining the effect of said agent on thelevel of the carboxyl-terminal apoE polypeptide in the cell, wherein anagent that reduces the level of the carboxyl-terminal truncated apoEpolypeptide is a candidate agent for modulating a phenomenon associatedwith AD.
 2. The method of claim 1, wherein the cell is a cell in anon-human transgenic animal that comprises, as a transgene, a nucleicacid that comprises a nucleotide sequence encoding apoE.
 3. The methodof claim 1, wherein the cell is an in vitro cell.
 4. The method of claim3, wherein the cell comprises a nucleic acid that comprises a nucleotidesequence that encodes the carboxyl-terminal truncated form of apoE. 5.The method of claim 1, wherein the apoE is apoE4.
 6. The method of claim5, wherein the carboxyl-terminal truncated form of apoE4 isapoE4(A272-299).
 7. The method of claim 3, wherein the cell is aneuronal cell.